Rhizobia transformants which symbiotically fixes nitrogen in non-legumes, a material for treating seeds of a non-legume plant, non-legume seeds, a non-legume plant and a method for producing rhizobia transconjungants

ABSTRACT

Rhizobia transformants that nodulate and fix nitrogen in non-legumes. The nodulated non-legume plants can be grown without nitrogenous fertilizer and have at least the same or higher protein content, dry matter content and nitrogen content than their non-nodulated counterparts which are fertilized by the addition of nitrogenous fertilizer. The straw remaining after harvesting the nodulated non-legumes is also high in protein content.

This is a continuation of application Ser. No. 07/133,107, filed Sep.28, 1987, now abandoned.

TABLE OF CONTENTS

1. Field of the Invention

2. Background of the Invention

2.1. Biological Nitrogen Fixation

2.1.1. Legume Genes Involved in Symbiosis

2.1.2. Rhizobium Genes Involved in Symbiosis

2.1.3. Biological Nitrogen Fixation and Non-Legumes

3. SUMMARY OF THE INVENTION

3.1. Definitions

4. Brief Description of the Figures

5. Detailed Description of the Invention

5.1. The Rhizobia Transformants

5.1.1. Nutrient Media for Identification of Rhizobia Transformants

5.1.2. The Alternating Line Culture Method for Producing RhizobiaTransconjugants

5.1.3. Alternate Methods for Producing Rhizobia Transformants

5.2. Non-Legume Seed Coatings and Coated Seeds

5.2.1. The Non-Legume Seed Coating

5.2.2. Methods for Coating the Non-Legume Seeds

5.3. Establishing Symbiotic Nitrogen Fixation in the Non-Legumes

5.4. The Nodulated Non-Legumes

5.4.1. Nodule Characteristics

6. Example: Materials and Methods for the Production of RhizobiaTransconjugants and Nodulation of Non-Legumes

6.1. The Alternating Line Culture Method

6.1.1. Isolation of Parent Rhizobia

6.1.2. Nutrient Agar Medium

6.1.3. Legume Plant Extract

6.1.4. Isolation of the Rhizobia Transconjugants

6.2. Preparation of the Non-Legume Seeds

6.3. Infection of Non-Legumes with Rhizobia F₂ Transconjugants

6.3.1. Laboratory Studies

6.3.2. Field Studies

6.4. Protocol for Analysis of Dry Mass, Nitrogen Content and ProteinContent of Nodulated Non-Legumes

7. Example:Rhizobium tritici which Nodulates Wheat

7.1. Preparation of Rhizobium tritici

7.2. Nodulation of Wheat with Rhizobium tritici

7.2.1. Laboratory Study

7.2.2. Field Study

7.3. Analysis of Dry Mass, Nitrogen Content and Protein Content ofNodulated Wheat

7.3.1. Results of Laboratory Study

7.3.2. Results of Field Study

8. Example:Rhizobium hordei which Nodulates Barley

8.1. Preparation of Rhizobium hordei

8.1.1. Characterization of Rhizobium hordei

8.2. Nodulation of Barley with Rhizobium hordei

8.2.1. Laboratory Study

8.3. Analysis of Dry Mass, Nitrogen Content and Protein Content ofNodulated Barley

8.3.1. Results of Laboratory Study

8.4. ¹⁵ N Enrichment in Nodulated Barley

8.5. Morphology of the Nodules

8.6. Antibiotic Resistance of Reisolated Barley Bacteroids

9. Example:Rhizobium sorghi which Nodulates Sorghum

9.1. Preparation of Rhizobium sorghi

9.2. Nodulation of Sorghum with Rhizobium sorghi

9.2.1. Laboratory Study

9.2.2. Field Study

9.3. Analysis of Nitrogen Content and Protein Content of NodulatedSorghum

9.3.1. Results of Laboratory Study

9.3.2. Results of Field Study

10. Example:Rhizobium oryzae Which Nodulates Rice

10.1. Preparation of Rhizobium oryzae

10.2. Nodulation of Rice With Rhizobium oryzae

10.2.1. Field Study

10.3. Analysis of Protein and Nitrogen Content of the Nodulated Rice

10.3.1. Results of Field Study

11. Example:Rhizobium as a Nitrogen Fertilizer for Eucalyptus

11.1. Preparation of Rhizobium eul

11.2. Treatment of Eucalyptus with Rhizobium eul

12. Example:Rhizobium Rl Which Nodulates Brassicas

12.1. Preparation of Rhizobium Rl

12.2. Treatment of Rape with Rhizobium Rl

13. Example: Total Nitrogen Analysis of Nodulated Non-Legumes

13.1. Materials and Methods

13.2. Wheat and Barley

13.3. Shorghum and Rice

13.4. Rape

14. Deposit of Microorganisms

1. FIELD OF THE INVENTION

The present invention discloses Rhizobia transformants that infect,nodulate and fix nitrogen in non-legume plants grown from seeds coatedwith a material specific for the Rhizobia transformant. The nodulatednon-legumes can be grown without using nitrogenous fertilizer; plantsand the straw remaining after harvest have a higher protein content, drymatter content and nitrogen content than their non-nodulatedcounterparts.

The invention is illustrated by way of specific examples in whichRhizobia transconjugants ar used to nodulate grasses (the Poaceaefamily) including wheat, barley, sorghum, rice and Brassicas (theCruciferae family) such as rape. Rhizobia transconjugants which fixnitrogen in eucalyptus (the Myrtaceae family) are also illustrated inthe examples.

2. BACKGROUND OF THE INVENTION

An essential aspect of plant metabolism is the use of nitrates and otherinorganic nitrogen compounds in the synthesis of organic compounds suchas amino acids, proteins, chlorophylls, vitamins, hormones and alkaloidswhich are essential to plant growth and development. Although plantsabsorb nitrates and other nitrogen compounds from the soil, the ultimatesource of nitrogen is the free dinitrogen (N₂) of the atmosphere;however, free dinitrogen as such, must be fixed and converted to a formthat can be utilized by the plant.

2.1. BIOLOGICAL NITROGEN FIXATION

Biological dinitrogen fixation (more commonly referred to as biologicalnitrogen fixation) is a complicated process involving the stepwisereduction of free nitrogen to ammonia through a series of intermediates;it is accomplished by nitrogen-fixing microorganisms some genera ofwhich live symbiotically with certain vascular plants. The mostimportant genus of the symbiotic nitrogen-fixing bacteria is Rhizobium,which has numerous species, each of which is symbiotic with one or a fewclosely related species of legume plants (e.g., peas, beans, clcvers,etc). As a result, legumes, unlike other plants which are unable to fixnitrogen, do not require nitrogenous fertilizers for growth; in fact,legumes can enrich the nitrogen content of the soil. In view of theeconomic importance of many legumes, bacterial cultures are availablefor inoculating legume seeds; in addition, legume seed coatingscontaining viable rhizobia have been disclosed (U.S. Pat. No. 3,499,748and U.S. Pat. No. 4,149,869).

Symbiotic nitrogen fixation involves the following: a Rhizobiumbacterium which is specific for a particular legume host infects theroot of the legume host. Thereafter a nodule develops on the root inwhich the Rhizobia live in an endosymbiotic state known as bacteroidswhich perform unique functions, such as nitrogen fixation, in closecooperation with the legume host; the bacteroid behaves almost like anorganelle. Interestingly, Rhizobia do not generally fix nitrogen explanta. For a review of various aspects of biological nitrogen fixationin legumes, see Chapters 3, 4, and 5 in "Plant Gene Research--GenesInvolved in Microbe Plant Interactions", D. P. S. Verma, T. H. Hohn,Springer-Verlag, N.Y., 1984.

Infection, nodulation and nitrogen fixation each involve an intricateinteraction between the Rhizobium and its specific legume host. In orderfor the initial infection to occur, the Rhizobium and the legume plantmust "recognize" each other. This very specific recognition is believedto involve an interaction between a lectin of the legume host (aglycoprotein found on the root hairs) and polysaccharides on thebacterial surface; this interaction can be regarded as a very specific"lock and key" type of mechanism, the specificity of which has beencompared to an antibody-antigen interaction, without which, infectioncannot occur. Following the specific recognition and attachment ofRhizobium to the root of a particular legume host, infection of the hostproceeds in a non-pathogenic fashion controlled by the host responses tothe invading Rhizobia. Generally, the Rhizobia enter the legume throughthe root hairs and bacterial invasion is mediated by a tubular structureformed by the host plant called the infection thread, which invades theinner cells of the root cortex. The Rhizobia are released from theinfection thread but remain enclosed in a host derived membrane envelopecalled the peribacteroid membrane; thus the bacteroids are restricted toextracellular compartments. Disruption of this delicate sequence ofevents could lead to infection that is pathogenic to the host.

After infection of the host legume by the Rhizobium, subsequentnodulation occurs only in response to an intricate interplay of eventsin which the legume influences the expression of Rhizobium genes and theRhizobium, in turn, influences activity of the legume genes required fornodulation. The cells in the nodule tissue are highly organized in zonesthat have plant-specific arrangements; the bacteria are routinely foundin the peribacteroid membranes which exclude the bacteria from theepidermal and meristematic zones. The organization and morphology of thenodule is significant biochemically in that problems of diffusion mustbe solved in order to provide for entry of oxygen and nutrients for thebacteroids and exit of ammonia for the host plant. A distinctive featureof effective root nodules, i.e., nodules which actually fix nitrogen, isthe presence of leghemoglobin, an oxygen binding protein which appearsto be involved in nodule respiration (i.e., the high concentration ofthis protein facilitates diffusion of oxygen into the bacteroid domains)and in the protection of nitrogenase (an enzyme essential in thenitrogen fixation pathway) from being poisoned by excess oxygen.Leghemoglogin is apparently a truly symbiotic product--the globinproteins are coded by plant genes and the haem synthesis is contributedby the bacteria

Ultimately, symbiotic nitrogen fixation which occurs in the nodule is ajoint endeavor The bacteroids contain the machinery for nitrogenfixation and the legume partner assimilates the ammonia produced into anorganic form which is then used for the nutrition of the whole plant andthe bacteroids; the legume partner provides a suitable environment and asource of energy that enables the bacteroids to fix nitrogen. Much cango wrong with the entire process: if the host plant bacteria are"mismatched" infection will not be successful, thus the whole process ofnodulation and symbiotic nitrogen fixation will not occur. Moreover,successful nodulation does not mean that nitrogen fixation willnecessarily occur because the resulting nodule could be defective.

2.1.1. LEGUME GENES INVOLVED IN SYMBIOSIS

Two major groups of host gene products, leghemoglobins and nodulins, areinduced specifically during symbiotic nitrogen fixation. While thestructure and function of leghemoglobin in the nodules is relativelyclear, little is known about the nodulins. Interestingly, a recentarticle reported that leghemoglobin plant DNA sequences were detectablein non-legumes, thus suggesting that the genes for these oxygen-bindingproteins may be more widely dispersed than previously thought (Hattori &Johnson, 1985, Plant Mol. Biol 4:285-292).

2.1.2. RHIZOBIUM GENES INVOLVED IN SYMBIOSIS

A few years ago it was discovered that Rhizobial enes needed fornodulation (nod) are located on a large plasmid (called the symbiosisplasmid) which also carries the nitrogen fixation (nif) genesthemselves. It has since been found that many of the Rhizobia bacteriacould be induced to recognize a new legume partner by transferring intothat rhizobium a symbiosis plasmid from another species of rhizobiumhaving a different host specificity. There is no indication thattransfer of Rhizobium symbiosis plasmids from one Rhizobium to anothercould expand the host range to non-legumes. Interestingly, theintroduction of a rhizobial symbiosis plasmid into Agrobacteriumtumefaciens (a bacteria which causes tumorous plant growth called crowngalls) confers on the Agrobacterium the ability to produce root nodulesin legumes; however the nodules are defective in that they do not fixnitroqen (Hooykaas, In "Molecular Genetics of the Bacteria-PlantInteraction", A. Puhler, eds, Springer-Verlag, NY, 1983, pp.229-239).

Over the years a variety of evidence has shown that nodulationefficiency of Rhizobia bacteria in legumes is increased by exposure tocertain plant exudates; perhaps this exposure stimulates expression ofthe nod gene. All legume root exudates produce this effect, but exudatesfrom a variety of non-legumes do not.

2.1.3. BIOLOGICAL NITROGEN FIXATION AND NON-LEGUMES

A non-rhizobium genus of bacteria called Frankia is symbiotic with somevascular non-legume plants such as Alnus (alder), Casuarina (cassowarytree), Ceanothus, Eleagnus, Myrica (myrtle) and Psychotria (a tropicalwoody plant). A report based upondata derived from in vitro associationsof a tobacco cell culture system and rhizobia, indicates that somenon-legumes may provide factors that can be utilized by Rhizobia (Gibsonet al., 1976, Planta 128: 233-239); however, no specific recognition,symbiotic relationship or specific interactions were indicated orobserved. In fact, the only known example, outside the legume order, ofa stable symbiosis between a non-legume seed plant and Rhizobium is thatof Parasponia, a tropical large Malayan (elm) family; naturallyoccurring Rhizobium strains isolated in Australia were found to nodulateand fix nitrogen in Parasponia (Trinick, 1980, New Phytol. 85:37-45 and86: 17-26; Trinick, 1981, Current Perspectives in Nitrogen Fixation,Gibson et al., eds. Elsevier Press, p.480; Trinick et al., 1976, Arch.Microbiol. 108:159-166; see also Bender et al., 1985, Plant Science38:138-140).

3. SUMMARY OF THE INVENTION

Rhizobia transformants are described which can symbiotically fixnitrogen in non-legumes. More specifically, the Rhizobium transformantsof the present invention can (a) infect the roots of non-legume plantsderived from seeds coated with a protein mixture specific for theRhizobium transformant: (b) nodulate the roots of these non-legumeplants: and (c) fix nitrogen in the nodules thus eliminating therequirement of using nitrogen fertilizer to promote plant growth anddevelopment in these non-legumes. A nutrient media is described which isuseful in the identification of various Rhizobia species and thetransformants of the present invention.

Coated non-legume seeds are also described which, in part, allow for theestablishment of symbiotic nitrogen fixation in non-legumes by theRhizobia transformants of the present invention. These seed coatingscomprise legume extracts, chromatographic fractions of legume extracts,crystals or purified proteins which are specific for the Rhizobiumtransformant. The seed coating may also include the Rhizobiumtransformant itself as an ingredient.

Methods are described for producing Rhizobium transformants, coating thenon-legume seeds and for establishing symbiosis between the Rhizobiumtransformants and the non-legume.

3 1. DEFINITIONS

As used herein, the term "Rhizobium transformant" is defined as aRhizobium containing introduced DNA which may be produced by any of anumber of methods, including but not limited to transformation (i.e.,infection with plasmid DNA), transfection (i.e., infection with freeDNA, phage DNA, or viral DNA), or conjugation (i.e., the transfer of areplica plasmid from one bacterium to another); the bacterialtransformants resulting from conjugation are a subset of thetransformants of the present invention and may be specifically referredto as transconjugants.

The term "parent Rhizobia", as used herein, is defined as the parentspecies which are conjugated to produce Rhizobium transconjugants of thepresent invention, as well as the Rhizobia species from which plasmidsor DNA sequences can be isolated and used to transform Rhizobia toproduce the Rhizobium transformants of the present invention.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph that represents the dry matter content (A) andnitrogen content (B) of wheat plants harvested at various times afterplanting. Three groups of wheat are represented: wheat nodulated byRhizobium transconjugants (+R-N); wheat fertilized with a nitrogenousfertilizer but not treated with Rhizobium transconjugants (-R+N); andwheat neither nodulated by Rhizobium transconjugants nor treated withnitrogenous fertilizer (-R-N).

FIG. 2 is a graph that represents the dry matter content (A) andnitrogen content (B) of barley plants harvested at various times afterplanting. Three groups of barley are represented: barley nodulated byRhizobium transconjugants (+R-N); barley fertilized with a nitrogenousfertilizer but not treated with Rhizobium transconjugants (-R+N); andbarley neither nodulated by Rhizobium transconjugants nor treated withnitrogenous fertilizer (-R-N).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to Rhizobia transformants thatnodulate and fix nitrogen in non-legumes. The nodulated non-legumeplants can be grown without nitrogenous fertilizer and have the same orhigher protein content, dry matter content and nitrogen content thantheir non-nodulated counterparts which are fertilized by the addition ofnitrogenous fertilizer. The straw remaining after harvesting thenodulated non-legumes is also high in protein content.

More specifically, the Rhizobia transformants of the present inventionare capable of nodulating non-legume plants grown from seeds coated witha material specific for the particular transformant. The invention isdirected to the Rhizobia transformants, methods for producing theRhizobia transformants, the material used to coat the non-legume seeds,the coated seeds themselves, methods for nodulating the non-legumeplants with the Rhizobia transformants, and the resulting nodulatednon-legumes which have a high protein content.

For purposes of clarity of description, the invention is discussed inthe subsections below in the following order: (a) the Rhizobiumtransformants; (b) the non-legume seed coatings; (c) establishingsymbiotic nitrogen fixation in the non-legume: and (d) the nodulatednon-legumes.

5.1. THE RHIZOBIA TRANSFORMANTS

The production and identification of the Rhizobia transformants of thepresent invention was based upon the initial discovery that eachdifferent species of Rhizobia, when grown outside its legume host onnutrient medium produces a colony having a particular color, providedthe nutrient medium contains, in addition to the nutrients necessary tosupport bacterial growth, a non-denatured extract derived from thelegume host which is the specific partner of the Rhizobium species. Thecolony color can be utilized as a means to identify the particularRhizobium species. In fact, different species of Rhizobia can becultured on a nutrient medium which contains a mixture of thenon-denatured extracts of each legume host specific for each species;each species will form colonies having its characteristic color.

The present invention is also based upon the further discovery that theRhizobia transformants which can infect, nodulate and fix nitrogen innon-legumes form snowy white colonies on nutrient media which containsextracts of the legume host partners of the parent Rhizobia of thetransformant; and that the transformant bacteroids which are isolatedfrom the non-legume root nodule and cultured on the medium defined abovewhich contains, in addition, an extract of the non-legume host, willform colonies that are greyish in color. Thus, the nutrient mediaprovide a relatively uncomplicated means for identifying thetransformants of the present invention. The nutrient media are describedin the subsection below.

5.1.1. NUTRIENT MEDIA FOR IDENTIFICATION OF RHIZOBIA TRANSFORMANTS

The nutrient medium should contain the nutrients necessary for growth ofthe Rhizobium, including but not limited to any of the well knownsources of carbon, nitrogen, and salts as well as vitamins of theB-group and the essential amino acids such as L-alanine, L-serine andL-tryptophan which can be present in the form of individual amino acids,tripeptides or oligopeptides, etc. The legume extract ingredient isuseful for the identification of Rhizobia species but is not necessaryfor the nutrition of the Rhizobia; i.e., if a Rhizobium is grown on anutrient medium that either does not contain its legume host extract orcontains denatured legume host extract (e.g., autoclaving the mediaafter the addition of the legume extract denatures the extract) colonieswill be formed but the colony of each Rhizobium species will be clear incolor. Table I below lists the characteristic colony colors formed bythe various Rhizobia species grown on nutrient medium containing theextract of the legume host partner of each species streaked on theplate. It should be noted that incubation of the medium used in theexamples of the present invention at a temperature above 32° C. willresult in the formation of red colonies by all Rhizobia colonies growingon the medium; this is a reversible color change in that thecharacteristic color of each colony will return when the temperature islowered, for example to somewhere between 18° C. and 30° C.Additionally, the nutrient medium can be altered, by the addition ofcystein and phenylalanine, to produce colonies having different shadesof the characteristic color for that species.

                  TABLE I                                                         ______________________________________                                        COLONY COLOR OF DIFFERENT RHIZOBIA SPECIES                                    GROWN ON NUTRIENT MEDIUM CONTAINING                                           EXTRACT OF THE HOST SPECIFIC LEGUME PARTNER*                                             Legume                                                             Rhizobium  Host Partner     Colony Color                                      ______________________________________                                        R. cowpea  Vigna (Peanut, Mimosa                                                                          greyish brown                                                Acacia, Leucaena)                                                  R. japonicum                                                                             Glycin (Soybean) reddish orange                                    R. leguminosarum                                                                         Lathyrus (Pea)   golden yellow                                     R. lupini  Lupinus (Lupine) light yellow                                      R. meliloti                                                                              Melilotus (Sweet Clover)                                                                       yellowish brown                                   R. phaseoli                                                                              Phaseolus (Bean) dark brown                                        R. trifoli Trifolium (Clover)                                                                             light brown                                       ______________________________________                                         *Colonies are incubated at a temperature below 32° C. (e.g.,           between 18° C. and 30° C.) on the nutrient medium described     in more detail in Section 6.1.                                           

The legume extracts used in the nutrient medium may be produced from anypart of the legume host plant, including but not limited to shoots,stems, roots or seeds; the extracts of young shoots appear to result inthe fastest color reaction. The legume extracts can be prepared bydividing the plant part into fine particles, homogenizing the maceratedmaterial in ethanol with a buffer at a pH of about 7.2 and pelleting theinsoluble material by centrifugation. The supernatant can be dialyzedagainst distilled water until it is clear whereupon it may be againcentrifuged. The entire process is carried out at 4° C. to minimizedecomposition of the plant substances.

The legume extract can be fractionated chromatographically according toa method which is a modification of Allen et al., 1973, Biochem. J. 131:155-162; Allen et al., February 1975, FEBS Letters 50(3): 362-364;Gordon et al., August 1972 FEBS Letters 24(2): 193-196; Peomans et al.,1982, Planta 156: 568-572; and Trowbridge, 1974, J. Biol. Chem. 249:6004-6012. Briefly, this involves the chromatographic separation of theextract using a column of galactose-derivatized CH-Sepharose 4B(Pharmacia, Sweden) and DEAE-52 (Whatman) as follows: (a) the extract isfirst applied to the galactose derivatized CH-Sepharose 4B column.Unbound substances are removed and saved by washing the column withbuffer and collecting 5 ml fractions which are assayedspectrophotometrically by absorbance at a wavelength of 280 μm; the washis continued until no significant absorbance is detected in theeffluent. The fractions which demonstrate absorbance at 280 nm arepooled and saved for chromatography on DEAE-52. The substances whichbound to the galactose-derivatized Sepharose 4B column bed are eluted bythe addition of 4% glucose to the column; the eluent is collected in 5ml fractions which are also assayed by absorbance at 280 nm; (b) thefractions containing the substances which did not bind to the galactosederivatized Sepharose 4B are applied to a DEAE-52 column, which is firsteluted at pH 7.2 and then at pH 9.2; five ml fractions are collected andassayed for absorbance at 280 nm. The fractions comprising the peakabsorbance value at pH 7.2 are pooled and those comprising a peakabsorbance value at pH 9.2 are pooled. These three fractions of aparticular legume host (i.e., the fraction eluted using glucose and thefractions eluted at pH 7.2 and p 9.2) can be combined and used in placeof the whole extract in the nutrient media described above. In fact, theactive component of the three fractions can be crystallized and stored.These crystals derived from the legume host can be used in place of thelegume host extract as an ingredient in the nutrient media used toidentify the Rhizobia transformants of the present invention.

Although we are not bound to any theory or explanation of the invention,the chromatographic fractions probably contain protein, and possiblyglycoproteins. The affinity to the derivatized Sepharose 4B suggeststhat it is likely that at least one of the proteins is a lectin. Thiscould be significant since legume lectins are thought to be important inthe initial recognition of Rhizobium partners. Once the amino acidsequences of these proteins are determined, these proteins can be madeby chemical synthetic methods or via recombinant DNA techniques, usingprokaryotic or eukaryotic host-vector expression systems to express theproteins. Expression of the protein in a eukaryotic host-vectorexpression system may be preferred because the eukaryote can process theprotein in a manner that is more similar to the naturally occurringproteins. This could be especially important if the protein is a lectin.In addition, the identified proteins may be isolated from sources otherthan the legume host. We do not, by this theory, exclude the possiblitythat other factors present in the extract such as carbohydrates,alkaloids, hormones, etc. could be a significant factor in the activityof the extract.

Although the nutrient media described above afford a convenient assayfor identifyinq the transformants of the present invention, apreliminary characterization of the transformant DNA has beenundertaken. The Rhizobia transformants of the present invention whichsymbioticially fix nitrogen in non-legumes appear to possess plasmidsthat are not contained in the parent Rhizobia of the transconjugant.While we are not bound to any theory or explanation of the invention, itis interesting to note that these plasmids could contain the DNAsequences responsible for the new host range of the Rhizobiumtransformants.

Methods for producing the Rhizobia transformants of the presentinvention are discussed in more detail below.

5.1.2. THE ALTERNATING LINE CULTURE METHOD FOR PRODUCING RHIZOBIATRANSCONJUGANTS

The alternating line culture method for producing Rhizobiumtransconjugants of the present invention involves the following: twodifferent Rhizobia species (the parent generation) are streaked inalternating rows on a solid nutrient medium containing in addition tothe nutrients essential for bacterial growth either: (a) a mixture ofnon-denatured extracts of the legume host partners of each Rhizobiaspecies; (b) the three chromatographic fractions obtained from theextract of each legume host as described in section 5.1.1. above; (c)the crystals obtained from the chromatographic fractions of each legumehost; or (d) proteins (indluding glycoproteins) related thereto.Hereinafter these components will be referred to as the legume extract,chromatographic fractions, crystals, or proteins. Each parent Rhizobiumspecies will form colonies having a characteristic color along thestreak. Rhizobia transconjugants (herein called the Rhizobia F₁transconjugants) are produced in between the alternating rows of parentcolonies. The Rhizobia F₁ transconjugants, unlike their parents, formmilky white colonies and cannot nodulate any plant.

The Rhizobia F₁ transconjugants are isolated from the milky whitecolonies and cultured in alternating rows with a third parent Rhizobiumspecies on solid nutrient medium containing in addition to the legumeextracts, chromatographic fractions, crystals or proteins used toproduce the F₁ transconjugant, a third non-denatured legume extract,chromatographic fractions, crystals or proteins derived from the legumehost partner of the third parent Rhizobium species. Rhizobium F₂transconjugants are produced in between the alternating rows of themilky white colonies formed by the Rhizobium F₁ transconjugant and thecolored colonies formed by the third parent Rhizobium. The Rhizobium F₂transconjugant colonies are identified by their snowy white color andare able to infect, nodulate and fix nitrogen in non-legumes grown fromseeds which are treated and sown as described herein.

The identification and selection of Rhizobium F₁ and F₂ transconjugantsproduced above is made possible because of the specilized media used.When each cross is performed, two kinds of colonies develop in betweenthe rows of parents: (a) colonies which have a color comprising amixture of the colors of the parent colonies and (b) the milky white F₁transconjugant colonies or the snowy white F₂ transconjugant colonies.The mixed colored colonies comprise Rhizobia which are not stable; theseRhizobia can nodulate the legume host partners of both parent Rhizobia,however, only in one generation. In other words, the bacteroidsrecovered from the nodules of each legume host can only nodulate thatparticular legume host again. The F₁ transconjugants of the milky whitecolonies are stable but are incapable of nodulating any plant.Surprisingly, the F₂ transconjugants of the snowy white colonies arestable Rhizobia which can nodulate and symbiotically fix nitrogen innon-legumes.

The parent Rhizobia used to produce the F₂ transconjugants can comprisea third species of Rhizobia, another F₁ transconjugant or possiblyanother F₂ transconjugant. If the third parent comprises another F₁transconjugant, then the nutrient media will contain at least fourlegume extracts, chromatographic fractions, crystals or proteins; i.e.,those of the legume host partner for each of the two parent Rhizobiaused to produce each of the two F₁ transconjugants. If the third parentcomprises an F₂ transconjugant, then the nutrient media will contain atleast five host legume extracts, chromatographic fractions, crystals, orproteins; i.e., those of the legume host partner for each of the twoparent Rhizobia used to produce the F₁ transconjugant as well as thelegume host partner for each of the three parent Rhizobia used toproduce the F₂ transconjugant.

The alternating rows of parent colonies is a convenient approach forproducing the transconjugants; however, any pattern or method ofinoculation may be used to grow the parent colonies in proximity to oneanother so that conjugation can occur. Thus circles, ellipses, wavy orspiral patterns may be used. In fact, a number of different parents canbe streaked on the plates to produce a number of differenttransconjugants on the same plate.

5.1.3. ALTERNATE METHODS FOR PRODUCING RHIZOBIA TRANSFORMANTS

Although the alternating line culture method is a convenient way toproduce Rhizobium F₂ transformants the present invention is not limitedto this method. In fact, in addition to conjugation, transformation withthe appropriate plasmid responsible for the increased host rangespecificity of the rhizobirum is contemplated as being within the scopeof the present invention. In addition, recombinant DNA techniques,including the use of phages and other vectors for infection of Rhizobiawith the appropriate sequences is contemplated as within the scope ofthe present invention.

For example, a particularly useful system for Rhizobia is the Tn5transposon which can be used in a scheme to transform Rhizobia.Symbiosis plasmids (Sym plasmids) can be identified by hybridizationwith a labeled DNA probe containing nif genes. Transfer of this symplasmid can be accomplished by using Tn5 to incorporate (a) a markergene into the sym plasmid, e.g., a gene for antibiotic resistance can beincorporated into the sym plasmid using transposon Tn5 by a conjugatingdonor and recipient bacteria and selecting hybrids based upon theacquired resistance; (b) by cloning the sym plasmid or portions thereofin E. coli and transforming Rhizobia with the plasmid itself; (c) byrecombining the plasmid with transport genes together with the markergene (e.q., antibiotic resistance) such as p^(vw5JI) or Tn5-Mob, forexample; and (d) by hybridizing the bacteria containing the plasmid withits incorporated marker gene with the recipient bacterium simultaneouslywith an auxiliary bacterium to promote transfer.

5 2. NON-LEGUME SEED COATINGS AND COATED SEEDS

In order to establish symbiosis between the Rhizobia transformants ofthe present invention, the seeds of the non-legume should be treated orcoated with a material specific for the Rhizobium transformants. Suchcoatings may include or exclude the Rhizobium transformant itself. Thepresence or absence of the Rhizobia transformant in the coating impactsonly on the method used to later establish symbiosis in the growingplant.

5.2.1. THE NON-LEGUME SEED COATING

The seed coating includes but is not limited to the following which mayincorporate or exclude the Rhizobium transformant: (a) a mixture ofextracts derived from each of the legume host plants which is a partnerto each parent of the Rhizobia transformant; (b) chromatographicfractions derived from each legume host plant which is a partner to eachparent Rhizobium of the transformant; (c) crystals derived from thelegume extract and/or its chromatographic fractions or (d) proteinsrelated thereto. As previously explained, the proteins can be producedvia chemical synthetic methods, recombinant DNA techniques or isolatedfrom alternate sources.

Although we are not bound to any theory or explanation of the invention,these seed coatings described above may contain any of a number offactors including but not limited to lectins, flovones, etc. which maybe important in establishing the symbiosis between the non-legume andthe Rhizobia transformant. Recently, expression of Rhizobium nodulingenes involved in normal root-hair curling, and, therefore, insymbiont/host recognition and nodulation, was reported to be induced bya legume root exudate (Rossen et al., 1985, EMBO 4(13A):3369-3373). Veryrecently, a group of compounds called flavones which are secreted fromlegume roots were reported to induce expression of nodulation genes inRhizobium (see Redmond et al., 1986, Nature 323: 632-635). Flavones arenormally produced in flowers and leaves of various plants, only legumesare known to secrete or contain flavones in roots. According to theRedmond et al. report, certain flavones in the root exudate from pea,bean, soybean, alfalfa and clover induced expression of the nodA gene inR. trifoli while root exudate from the non-legumes maize, rice andParasponia did not. The stimulation of the Rhizobium was not legume-hostspecific from a symbiotic point of view; i.e., the expression of nodAwas induced in the Rhizobium in response to a number of different legumeexudates. Therefore, the flavones may be one of the early signalsbetween a Rhizobium and a legume after which a second more host specificrecognition takes place (for example, the lectin-polysacchariderecognition system) to establish that particular host-symbiontpartnership. Possibly the flavone induction system is an early eventwhich enables the non-legume seeds treated with legume extracts tobecome receptive to infection with the F₂ Rhizobium transformants of theinvention. Perhaps flavones present in the legume extracts are taken upby the non-legume seed during the imbibition or pregermination,transported to the root of the young seedling and when exuded attractthe Rhizobium transformant.

Other factors from the legume extracts contained in the seed coatingsherein may be important in the establishment of the non-legume host F₂transconjugant infection. For example, the seed coatings may containlectins which are believed to be involved in the specific recognitioninteraction between Rhizobia and their legume host partners. A recentreport describes a particulate form of the lectin, Trifolin A, which canbe isolated from clover root exudates and which specifically binds to R.trifoli (Truchet et al , 1986, Physiol. Plant 66: 575-582).Interestingly, legume root exudates containing lectin restored theability of mutant Rhizobia to recognize and nodulate their legume host(see Halverson et al., 1984, Plant Physiol. 74: 84-89 and 1985, PlantPhysiol. 77: 621 -625). Quite possibly one or more lectins are presentin the seed coatings of the invention which enable the specificrecognition between the Rhizobia transformant and its non-partner totake place.

5 2 2. METHODS FOR COATING THE NON-LEGUME SEEDS

The non-legume seeds may be coated by a variety of methods including butnot limited to immersion and air drying, spraying, encapsulation (forexample in polymers), immersion and drum drying, etc. The method chosenwould depend upon the coating to be used. For example, if the Rhizobiatransformant is included in the coating mixture, methods or ingredientswhich ensure the viability of the bacteria should be used; these includebut are not limited to: coating mixtures which contain a gel material tokeep the moisture levels high; alternatively, the seeds can be coatedwith pulverized seed husks which contain fungicides. In any case, themethod should be accomplished so that the active materials in thecoating are not denatured or destroyed.

In the examples of the present invention, the non-legume seeds werecoated by immersion in the legume extracts and air drying; preferablythree times. Two immersions resulted in poor penetration of the Rhizobiatransformants, whereas four immersions did not seem to improvepenetration.

5.3. ESTABLISHING SYMBIOTIC NITROGEN FIXATION IN THE NON-LEGUMES

A number of methods can be used to ensure infection of the non-legume bythe Rhizobia transformants; the method chosen depends, in part, upon thenature of the non-legume seed coating used.

For example, non-legume seeds treated as described with a coating thatdoes not contain the Rhizobium transformants can be watered with asuspension of the Rhizobium transformant. Thus the seed, seedling orplant can be watered with an appropriate volume of the bacterialsuspension. Where the seed coating contains the Rhizobium transformant,only planting and watering are required.

It may be desirable to add non-nitrogenous fertilizers depending uponthe type of plant, condition of the soil or terrain, etc. In fact, inthe laboratory experiments we have found that the addition of 2 mMnitrogen at the initial sowing of the seeds is enough to increase theinitial growth of the plant without harming or "shutting off" theRhizobia transconjugants. This more closely simulates the naturalcondition since most soil (even unfertilized) contains some amount ofnitrogen (no more that 2 mM); the addition of nitrogen in the field isnot required.

5 4. THE NODULATED NON-LEGUMES

The non-legumes that are nodulated by the Rhizobia transformants of thepresent invention have a nitrogen content and dry matter content that isequal to or greater than their non-nodulated counterparts fertilizedwith nitrogen; the nodulated plants have a higher nitrogen content anddry matter content than their unfertilized non-nodulated counterparts.Analyses of the amino acid compositions of the nodulated plants revealedthat in most cases the proportion of each amino acid remains the samebut the total concentration per plant is increased. However, in somecases, higher levels of tryptophan and leucine have been observed.

Interestingly, we have found that the straw remaining after harvest ofsome of the nodulated non-legume species consistently had a proteincontent of about 6% to about 9%; this is in striking contrast to theprotein content of 1.5% to 2% normally found in the straw of thenon-nodulated counterparts. This high protein straw can advantageouslybe used as a protein source, for example, in animal feed mixtures forboth farm and domestic animals.

The Rhizobium transformants of the present invention which nodulate andfix nitrogen in non-legumes can reduce the use of costly nitrogenousfertilizer and ultimately can improve the soil.

5.4.1. NODULE CHARACTERISTICS

The morphology of the non-legume nodules formed by the Rhizobiatransformants of the present invention is quite normal in appearance,however, a larger proportion of smaller nodules are formed. The Rhizobiaappear to enter the root via infection threads and the bacteroids seemto be maintained in compartments. In fact, a reddish color is observedin the nodule which may be leghemoglobin or a protein closely relatedthereto. Electron microscopy of the nodules revelas that the vascularbundles found in the cortex are peripherally located, whereas thevascular bundles of lateral roots are centrally located. Most of thecells in the central portion of the nodules are filled with bacteroids.

6. EXAMPLE:MATERIALS AND METHODS FOR THE PRODUCTION OF RHIZOBIATRANSCONJUGANTS AND NODULATION OF NON-LEGUMES

The examples that follow describe the nodulation by Rhizobiumtransformants of the present invention of the following non-legumesbelonging to the grass family (Poaceae): wheat, barley, sorghum andrice. In addition, a member of the Brassicas, a plant genus outside thegrass family (i.e., the Cruciferae family) is also nodulated by aRhizobium transformant of the present invention. Interestingly, apositive effect was observed on another plant outside the grass family,Eucalyptus (a member of the Myrtaceae family). In each example, thealternating line culture method was used to produce Rhizobiumtransconjugants. The non-legume seeds were coated by immersion of theseeds in legume extracts specific for the particular Rhizobiumtransconjugant used to nodulate the non-legume. The coated non-legumeseeds were infected with the Rhizobia transconjugants by sowing thecoated seeds, allowing them to germinate and watering the seedlings witha suspension of the Rhizobium transconjugant; nitrogen-fixing nodulesdeveloped in 8-12 weeks.

In the examples, laboratory studies were conducted in which thenon-legumes were divided into three groups treated as follows: (a) thefirst group was treated with the Rhizobium transconjugant withoutnitrogenous fertilizer (+R-N); (b) the second group was treated withnitrogenous fertilizer without the Rhizobium transconjugant (-R+N); and(c) the third group was treated with neither the Rhizobiumtransconjugant nor nitrogenous fertilizer (-R-N). At certain timesduring growth, the plants were sampled and the total content of organicnitrogen per plant, the amount of dry matter per plant, and in someexperiments the protein content per plant and its amino acid compositionwere determined. In some examples, field studies were conducted. Theresults demonstrated that, in each case, the non-legume plants nodulatedby the Rhizobia transconjugants were able to fix nitrogen and did notrequire nitrogenous fertilizer for growth. In fact, in many cases, thenodulated non-legumes (+R-N) had a higher nitrogen content and drymatter content than either the fertilized non-nodulated group (-R+N) orthe untreated group (-R-N).

Unless otherwise indicated, the materials and methods described belowwere used in each example that follows.

6.1. THE ALTERNATING LINE CULTURE METHOD

The alternating line culture method used in each example that followsinvolves the following: two different Rhizobia species (the parentgeneration) were streaked in alternating rows (3 mm apart) on a nutrientagar medium containing, in addition to nutrients necessary for growth, anon-denatured extract of each legume host specific for each parentRhizobium. After incubation at a growth temperature below 32° C., e.g.,between 18° C. and 30° C., each parent Rhizobium formed colonies havinga color characteristic for that species; Rhizobia transconjugants (F₁transconjugants) were produced between the alternating rows of thecolored parent colonies. The Rhizobia F₁ transconjugants unlike theirparents, formed milky white colonies and could not nodulate plants. Itshould be noted that incubation at a temperature above 32° C. on theagar medium used herein results in the formation of red colonies by allRhizobia species streaked on the plate; however, if the temperature islowered to a temperature below 32° C. (e.g., preferably between 18° C.to 30° C.) the characteristic color of each colony will reappear.

The Rhizobia F₁ transconjugants were isolated from the milky whitecolonies and cultured as described in alternating rows (3 mm apart) witha third Rhizobium parent species on nutrient agar medium containing, inaddition to the non-denatured legume extracts used to produce the milkywhite colonies, a third non-denatured legume extract derived from thelegume host specific for the third parent Rhizobium species. Rhizobia F₂transconjugants were produced in between the alternating rows of themilky white colonies formed by the Rhizobium F₁ transconjugant and thecolored colonies formed by the third parent Rhizobium. The Rhizobium F₂transconjugant colonies were identified by their snowy white color;these F₂ transconjugants were able to infect, nodulate and fix nitrogenin non-legumes which were treated and planted as described. Thematerials and methods used to produce the Rhizobium transconjugants aredescribed in more detail below.

6 1.1. ISOLATION OF PARENT RHIZOBIA

The Rhizobia species used as the parent generations were isolated eitherfrom soil samples or from legume nodules which were at an advanced stageof development.

Isolation of parent Rhizobia from the soil was accomplished by placingseeds of host legume plant which are nodulated by the Rhizobia in thesoil samples. The plants were harvested after 3-4 weeks (normally) or10-12 weeks if slow-growing.

Isolation of parent Rhizobia from legume nodules was accomplished asfollows: the nodules were severed from the roots of the legume plantalong with approximately 1 cm of the root tissue surrounding the nodulewhich was sterilized by immersion in 3% HgCl₂ and rinsed in 80% ethanol.The nodules were then excised from the root tissue and crushed in amortar under aseptic conditions. The macerated material was spread onthe nutrient agar medium described below. The colonies were purified bysubculturing on a medium of the same composition until single colonieswere formed twice. The Rhizobia isolated from the purified singlecolonies were streaked in alternating rows on the nutrient agar mediumdescribed in order to culture the alternating rows of parent Rhizobiacolonies.

6.1.2. NUTRIENT AGAR MEDIUM

The agar medium used to culture the Rhizobia in alternating lines wasprepared by adding the following nutrients in the amounts indicted to100 ml distilled water:

    ______________________________________                                        KH.sub.2 PO.sub.4      0.15 g                                                 K.sub.2 HPO.sub.4      0.15 g                                                 KNO.sub.3              2.50 g                                                 (NH.sub.4).sub.2 SO.sub.4                                                                            0.135 g                                                MgSO.sub.4.7H.sub.2 O  0.25 g                                                 Mannitol              10.00 g                                                 Agar                  12.00 g                                                 Solution No. 1         1.00 ml                                                Yeast extract          5.00 ml                                                ______________________________________                                    

Solution No. 1 had the following composition:

    ______________________________________                                        MnCl.sub.2.4H.sub.2 O                                                                         500 mg                                                        H.sub.3 BO.sub.3                                                                              300 mg                                                        ZnSO.sub.4.2H.sub.2 O                                                                         200 mg                                                        NaMoO.sub.4.2H.sub.2 O                                                                         20 mg                                                        CuSO.sub.4.5H.sub.2 O                                                                          2 mg                                                         CoCl.sub.2.6H.sub.2 O                                                                          2 mg                                                         Distilled water to a final volume of 100 ml                                   ______________________________________                                    

The agar medium defined above was then autoclaved for 20 minutes at 2bar and cooled to a temperature between 55° C. and 60° C. upon which thefollowing was added:1.0 ml of each Solution No. 2, Solution No. 3 andSolution No. 4; 20 ml each of non-denatured legume plant extractsderived from the host legume plant specific for each parent Rhizobia tobe cultured on the media; and 15 mg of each of the following aminoacids: L-alanine, L-serine and L-tryptophan. To avoid denaturing theproteins in the legume extract the final media composition should not beautoclaved.

Solution No. 2, Solution No. 3 and Solution No. 4, had the compositionsindicated below. Each was prepared using sterile distilled water.

    ______________________________________                                        Solution No. 2:                                                               Nicotinic acid    50 mg                                                       Thiamine HCl      50 mg                                                       Pyridoxin HCl     50 mg                                                       Myoinositol       50 mg                                                       Biotin            50 mg                                                       Sterile distilled water to final volume of 100 ml                             Solution No. 3:                                                               Potassium Iodate  75 mg                                                       Sterile distilled water                                                                         100 ml                                                      Solution No. 4:                                                               CaCl.sub.2.H.sub.2 O                                                                            15 gm                                                       Sterile distilled water                                                                         100 ml                                                      ______________________________________                                    

6.1.3. LEGUME PLANT EXTRACT

The legume plant extracts used in the agar medium and for coating thenon-legume seeds were prepared as described below.

The whole plant, or selected plant parts, e.g. clean washed roots,sterilized seeds or aerial parts, or preferably, young shoots, weredivided into fine particles, ground in a mortar with 80% ethanol to athink, homogeneous paste into which was added an equal volume ofpotassium-sodium salt buffer, pH 7.2, comprising the following:

    ______________________________________                                        K.sub.2 HPO.sub.4                                                                         0.430 g                                                           NaH.sub.2 PO.sub.4                                                                        1.469 g                                                           NaCl        7.200 g                                                           Sterile distilled water to a final volume of 1000 ml                          ______________________________________                                    

The mixture of the homogenized plant extract and buffer was allowed torest for 48 hours at 4° C. and was pelleted by centrifugation atapproximately 5000× g for 30 minutes. Then, the supernatant was dialyzedagainst sterile distilled water at 4° C. for about 48 hours, duringwhich time the water was replaced 5 to 6 times, until a clear, colorlessliquid, i.e., the extract, was obtained. The extracts were stored at 4°C.

6.1.4. ISOLATION OF RHIZOBIA TRANSCONJUGANTS

When the two parent Rhizobia were cultured at a temperature below 32° C.in alternating lines (3 mm apart) on the agar medium containing theappropriate legume extracts as described above, the parent Rhizobiaformed colonies having a characteristic color. However, between the rowsof colored parent colonies two types of colonies developed: (a) colonieshaving a color which is a mixture of the colors of the parent coloniesand (b) the milky white colonies of the Rhizobium F₁ transconjugant. Theproportion between the colored colonies and the F₁ transconjugant milkywhite colonies varied somewhat but on the average ranged from about100:3 (mixed colored colonies: milky white colonies). The mixed coloredcolonies nodulated both host legume species, but only in one generation;i.e., the Rhizobia of the mixed colored colonies nodulated the legumehost partners of both parent Rhizobia but the Rhizobia recovered fromthe nodules of each legume host could only nodulate that particularlegume host again. By contrast, the F₁ transconjugant milky whitecolonies do not nodulate any plant.

Since it was difficult to transfer a single hybrid colony to a newmedium without polluting it with one or more colored colonies, abiological cleaning was performed one or more times by transferring themilky white colonies to another agar medium (of the same composition)until, typically after 10 to 12 weeks, a pure culture of the F₁transconjugant with a stable and uniform milky white colour wasobtained.

The pure culture of F₁ transconjugant which formed the milky whitecolonies was then cultured at a temperature below 32° C. in alternatinglines (3 mm apart) with another Rhizobium species on an agar medium ofthe same composition used for the F₁ transconjugant, which contained inaddition, a third legume extract derived from the legume host specificfor the third parent Rhizobium. The F₁ transconjugant produced itscharacteristic milky white colonies whereas the parent Rhizobiumproduced colonies having its specific color. Two types of coloniesdeveloped between the lines of milky white colonies and the coloredcolonies: colored colonies and snowy white colonies. The snowy whitecolonies comprise the Rhizobium F₂ transconjugants which can formnitrogen fixing nodules in non-legumes. The F₂ transconjugant snowywhite colonies were cleaned and isolated as described for the milkywhite colonies and were used to nodulate non-legumes.

After nodulation of the non-legume plant, the Rhizobia F₂ transconjugantbacteroids can be isolated from the nodule and cultured in an agarmedium. If the agar medium has the same composition as the agar mediumused to produce the transconjugant (i.e., the agar medium contains thethree legume extracts derived from the legume host specific for eachparent Rhizobium used to produce the F₂ transconjugant) plus an extractof its non-legume host plant, then the F₂ transconjugants isolated fromthe non-legume nodule will form colonies having a greyish color.

6.2. PREPARATION OF THE NON-LEGUME SEEDS

Seeds of the non-legume plant were coated by immersion three times at20° C. for 3 hours each time in an aqueous solution containing 3%calcium sulphate, and up to 10% a mixture of the three legume extracts,prepared as described in Section 6.1.3., which were used to produce theRhizobium F₂ transconjugant. After each immersion the seeds were airdried at 40° C. for 12 hours.

6.3. INFECTION OF NON-LEGUMES WITH RHIZOBIA F₂ TRANSCONJUGANTS

Infection of non-legumes with Rhizobia F₂ transconjugants wasaccomplished by sowing the coated non-legume seeds and watering thegerminated seedlings with a suspension of the Rhizobium F₂transconjugant. In the examples that follow, laboratory studies andfield studies were conducted in which the germinated seedlings weretreated with either (a) the Rhizobium F₂ transconjugant withoutinorganic nitrogen fertilizer (+R-N); (b) inorganic nitrogen fertilizerwithout the Rhizobium F₂ transconjugant (-R+N); or (c) neither theRhizobium F₂ transconjugant nor the inorganic nitrogen fertilizer(-R-N). The nitrogen content per plant, the dry weight and in some casesthe amino acid composition of the resulting plants were determined.

The materials and methods used are described in greater detail below.

6.3.1. LABORATORY STUDIES

In the laboratory studies, the coated non-legume seeds were sown in oneliter containers (Jydsk Papir Vaerk, Arhus) filled with 3mm"Fibo"(R)-clinkers; 4 to 5 seeds per container were sown. The "Fibo"clinkers are air filled, burnt clay pebbles of approximately 3 mm indiameter which are normally used as isolation material.

When the germinated seeds grew a few centimeters above the "Fibo"clinker surface the seedlings in each container were watered as follows:(a) the +R-N group were watered with a 50 ml suspension of the RhizobiumF₂ transconjugant; thereafter the plants were watered once a week with50-80 ml per container of a non-nitrogenous fertilizer; (b) the -R+Ngroup were not treated with the Rhizobium F₂ transconjugant but insteadwere watered with an inorganic nitrogen fertilizer; thereafter theplants were watered once a week with 50-80 ml per container of the sameinorganic nitrogen fertilizer; and (c) the -R-N group were watered with50-80 ml per container of the non-nitrogenous fertilizer. The inorganicnitrogen fertilizer and non-nitrogenous fertilizer used are definedbelow.

    ______________________________________                                                       Fertilizer ml                                                                 Stock Soln/liter Final Volume                                  STOCK SOLUTIONS  Nitrogenous                                                                              Non-Nitrogenous                                   ______________________________________                                        NH.sub.4 NO.sub.3                                                                       (1.0 M)    20          0                                            CaSO.sub.4.2H.sub.2 O                                                                   (0.012 M)  65         65                                            KH.sub.2 PO.sub.4                                                                       (0.10 M)   20         20                                            MgSO.sub.4.7H.sub.2 O                                                                   (0.20 M)   15         15                                            Fe(EDTA):                                                                     FeSO.sub.4.7H.sub.2 O                                                                   (2.490 g)  10         10                                            Na.sub.2 EDTA                                                                           (3.716 g)                                                           Final Volume 1 liter                                                          MnCl.sub.3.4H.sub.2 O                                                                   (106.1 mg/l)                                                                             10         10                                            H.sub.3 BO.sub.3                                                                        (142.2 mg/l)                                                                             10         10                                            ZnSO.sub.4.7H.sub.2 O                                                                   (110.7 mg/l)                                                                             10         10                                            CuSO.sub.4.5H.sub.2 O                                                                   (8.0 mg/l) 10         10                                            Na.sub.2 MoO.sub.4.2H.sub.2 O                                                           (11.1 mg/l)                                                                              10         10                                            ______________________________________                                    

The suspensions of Rhizobia F₂ transconjugants which were used to waterthe seedlings were produced by culturing the Rhizobium F₂ transconjugantin 300 ml flasks containing 200 ml of the following nutrient medium:

    ______________________________________                                        KH.sub.2 PO.sub.4       1.0 g                                                 K.sub.2 HPO.sub.4       1.0 g                                                 MgSO.sub.4.7H.sub.2 O   0.36 g                                                CaSO.sub.4.2H.sub.2 O   0.17 g                                                FeCl.sub.3.6H.sub.2     0.005 g                                               KNO.sub.3               0.7 g                                                 Yeast extract           1.0 g                                                 Mannitol                3.0 g                                                 Distilled water to a final volume of 1000 ml                                  ______________________________________                                    

The bacteria were grown at 28° C. for two to three days until thebacterial density of the culture measured spectrophotometrically atOD₆₂₀ was 0.8, thus indicating that the bacteria were in the logarithmicgrowth phase and the culture had not entered the stationary phase. Thenthe cells were pelleted by centrifugation at approximately 5,000× g for30 minutes and washed by resuspension in sterile water. This wash wasrepeated once or twice in order to remove all nitrogenous compounds fromthe bacterial cells. The final pellet of cells was resuspended in 1.8liter sterile water; this final bacterial suspension was used to waterthe non-legume seedlings.

6.3.2. FIELD STUDIES

In the field studies, the coated non-legume seeds were sown in soil thathad never been cultivated. The land was cleared and divided into stripsthat were 30 feet wide by 90 feet long. The seeds were sown only inalternate strips (i.e., strips of unsown land remained in between thestrips where the seeds were sown) in order to prevent chemicals leachingout of one strip from entering and contaminating another strip. Within astrip, the seeds were sown in six rows; each row was separated by 60inches and the seeds within each row were sown 8 inches apart.

Each strip contained one of the following groups: (a) the +R-N groupwhich was treated with the Rhizobium F₂ transconjugant and watered withthe previously defined non-nitrogenous fertilizer, (b) the -R+N groupwhich was not treated with the Rhizobium F₂ transconjugant but insteadwas watered with the previously defined inorganic nitrogen fertilizer;and (c) the -R-N group which was neither treated with the Rhizobium F₂transconjugant nor the inorganic nitrogen fertilizer but instead waswatered with the non-nitrogenous fertilizer.

The following procedure was used to infect the +R-N group with theRhizobium F₂ transconjugant: a culture of the Rhizobium F₂transconjugant was inoculated 4 inches below each seed that was sown.The sowing and inoculation of the soil was accomplished by a machinewhich had a 300 gallon tank containing a suspension of the Rhizobia F₂transconjugant prepared as previously described on a much larger scale(i.e., the Rhizobia F₂ transconjugant was grown to log phase in a 10,000gallon tank and diluted to an O.D.₆₂₀ of 0.8). Approximately 1.5 ml/seedof the Rhizobium F₂ transconjugant culture was inoculated into theunderlying soil. Thereafter the seeds and seedlings were watered asdescribed.

6.4. PROTOCOL FOR ANALYSIS OF DRY MASS, NITROGEN CONTENT AND PROTEINCONTENT OF NODULATED NON-LEGUMES

Periodically a number of containers from each plant group in thelaboratory studies were thinned out to 3 plants per container andremoved for Kjeldahl-analysis in which the total content of organicnitrogen and the amount of dry matter per container (i.e., per 3 plants)was determined. To this end, the aerial parts of the plants wereremoved, dried for 48 hours at 80° C., weighed, crushed and ground in amortar. Samples of the macerated material were removed for theKjeldahl-analysis, which was carried out according to the standardmethod. Each Kjeldahl-analysis was repeated from 4 to 6 times. Theresults were recorded in terms of weight of nitrogen and weight of drymatter per plant. In all cases the Rhizobium nodulated plants had ahigher content of dry matter and nitrogen than did the nitrogenfertilized or untreated groups.

In the field studies, a number of plants were harvested periodically andthe protein content per plant was determined by first assaying thenitrogen content per plant using the Kjeldahl analysis and multiplyingthe percent nitrogen by a factor of 6.25. In some studies, the aminoacid composition of the protein was determined. In all cases theRhizobium nodulated non-legumes had a higher protein content than thatof the plants which were treated with the inorganic nitrogen fertilizerand the plants which were treated with the non-nitrogenous fertilizer.

7. EXAMPLE: RHIZOBIUM TRITICI WHICH NODULATES WHEAT

Rhizobium tritici, which nodulates wheat, was produced according to themethod described in Section 6 by first crossing R. phaseoli with R.cowpea leucaena to produce the F₁ transconjugant which was then crossedwith R. trifoli. The R. tritici thus produced was used to nodulate fourtypes of wheat: Anja, Kraka, Vuka and Williams.

7.1. PREPARATION OF RHIZOBIUM TRITICI

For the production of R. tritici the following parent Rhizobia were usedto produce the F₁ transconjugant milky white colonies: (a) R. phaseoli,isolated from a kidney bean cultivar, Prospector, grown in a soil samplefound near Aarhus, MS-1; and (b) R. cowpea leucaena, isolated from thetropical tree Leucaena leucocephala (belonging to the family ofFabaceae) from Papua, New Guinea.

The parent Rhizobia were cultured in alternating lines on the agarmedium previously described; the legume extracts used in the medium werederived from the aerial parts of the kidney bean and from the leaves ofthe Leucaena leucocephala. respectively. The colonies formed by R.phaseoli had a characteristic dark brown color whereas the coloniesformed by R. cowpea leucaena had a characteristic greyish brown color.

The Rhizobium F₁ transconjugant derived from the milky white colonieswas cleaned and cultured as described in alternating lines with a strainof R. trifoli which was isolated from red clover, grown in a soil samplefound in the vicinity of Randers. The legume extracts used in the mediumwere derived from the aerial parts of the kidney bean, the leaves ofLeucaena leucocephala and the aerial parts of red clover. The coloniesformed by the F ₁ transconjugant were milky white whereas the coloniesformed by R. trifoli had a characteristic light brown color.

The Rhizobium F₂ transconjugant derived from the snowy white coloniesobtained in between the streaks, herein called Rhizobium tritici, wascleaned and isolated as described; this took approximately 20 weeks, andthen the F₂ transconjugant was used to nodulate wheat.

7.2. NODULATION OF WHEAT WITH RHIZOBIUM TRITICI

The Rhizobium tritici was used to nodulate four types of wheat: Anja,Kraka, Vuka and Williams. The wheat seeds were treated by immersionthree times in the aqueous solution previously described containing 3%calcium sulphate and up to approximately 10% legume extracts which wereused to produce the R. tritici; i.e., the three legume extracts used tocoat the seeds were derived from the aerial parts of the kidney bean,the leaves of Leucaena leucocephala and the aerial parts of red clover.After each immersion the seeds were air-dried and sown as describedbelow.

7.2.1. LABORATORY STUDY

The experimental protocol previously described was carried out for threetypes of wheat used: Anja, Kraka and Vuka; i.e., 4 to 5 coated seedswere sown per one liter container filled with "Fibo"-clinkers andallowed to germinate. Each of the three types of wheat was divided intothe following three groups; (a) +R-N, the seedlings of which werewatered with a 50 ml suspension of R. tritici prepared as previouslydescribed, followed by weekly watering with the non-nitrogenousfertilizer; (b) -R+N, the seedlings of which received no R. tritici butwere watered with the inorganic nitrogen fertilizer; and (c) -R-N, theseedlings of which received neither the R. tritici nor inorganicnitrogen fertilizer and instead were watered with the non-nitrogenousfertilizer previously described. The plants treated with R. triticideveloped root nodules in 8 to 10 weeks. The plants in each containerwere thinned to three plants, and ten containers were used in each groupfor analysis of dry mass and nitrogen content.

7.2.2. FIELD STUDY

For the field study, the coated wheat seeds were sown as described inSection 6.3.2. The seeds of lot (a) +R-N, were treated as described witha suspension of R. tritici and were watered with non-nitrogenousfertilizer; those of lot (b) -R+N, were not treated with R. tritici butwere watered with the inorganic nitrogen fertilizer; and those of lot(c) -R-N, were not treated with either R. tritici or the inorganicnitrogen fertilizer, and were simply watered with the non-nitrogenousfertilizer.

7.3. ANALYSIS OF DRY MASS, NITROGEN CONTENT AND PROTEIN CONTENT OF THENODULATED WHEAT

The analytical methods described in Section 6.4 were used tocharacterize the nodulated wheat plants.

7.3.1. RESULTS OF LABORATORY STUDY

The wheat plants were harvested from the containers at 56, 70, 87, 100and 118 days after sowing. The dry weight per plant and nitrogen contentper plant (Kjeldahl analysis) were analyzed as previously described.

The results are shown in FIG. 1 in which the dry weight per plant (A)and nitrogen content per plant (B) are plotted over the number of daysafter sowing.

In order to facilitate the understanding of the figure, the followinginformation about the average weight of dry matter and average contentof nitrogen for the three wheat types is helpful:

    ______________________________________                                                   Dry matter    Nitrogen                                             Wheat      grams per 100 seeds                                                                         mg per seed                                          ______________________________________                                        Anja       4.403         1.103                                                Kraka      3.611         0.896                                                Vuka       4.052         0.819                                                ______________________________________                                    

The number of harvested plants at the various time points were:

    ______________________________________                                                 Number of Plants Harvested                                                    Days After Sowing                                                    Plant Group                                                                              56        70    87     100  118                                    ______________________________________                                        Trial One:                                                                    +R-N       33        30    21     23   25                                     -R+N       24        30    20     21   20                                     -R-N       24        26    22     26   35                                     Trial Two:                                                                    +R-N       11        10     7      8    8                                     -R+N        8        10     7      7    7                                     -R-N        8         9     7      9   12                                     ______________________________________                                    

The results shown in FIG. 1 demonstrate that during the growth periodthe Rhizobium treated wheat plants had a higher content of nitrogen anddry matter than the plants which were fertilized with nitrogen; at thetermination of the experiment the dry content of the +R-N plants wasalmost 40% higher than that of the -R+N plants and both of thesecategories had a higher weight than the untreated -R-N wheat plants.

7.3.2. RESULTS OF FIELD STUDY

The wheat plants in the field study were harvested after one season ofgrowth and the protein content per plant was analyzed as previouslydescribed. The results shown in Table II clearly demonstrate that theplants treated with R. tritici (+R-N) had a higher protein content thaneither the group treated with inorganic nitrogen fertilizer (-R+N) orthe group treated with non-nitrogenous fertilizer (-R-N).

                  TABLE II                                                        ______________________________________                                        PROTEIN CONTENT OF WHEAT                                                                    Protein Content                                                 Plant Group   per Plant                                                       ______________________________________                                        +R-N          22-28%                                                          -R+N          12-14%                                                          -R-N          ND*                                                             ______________________________________                                         * ND; No data; these untreated plants died after 8 weeks.                

8. EXAMPLE: RHIZOBIUM HORDEI WHICH NODULATES BARLEY

Rhizobium hordei which nodulates barley, was produced according to themethod described in Section 6 by first crossing R. phaseoli with R.leguminosarum to produce the F₁ transconjugant which was then crossedwith R. cowpea leucaena. The R. hordei thus produced was used tonodulate four types of barley: Hasso, Cerise, Harry and Igri.

8.1 PREPARATION OF RHIZOBIUM HORDEI

For the production of R. hordei the following parent Rhizobia were usedto produce the F₁ transconjugant milky white colonies: (a) R. phaseoli,isolated from a kidney been cultivar, Prospector, grown in a soil samplefound near Aarhus; and (b) R. lequminosarum, isolated from garden pea.

The parent Rhizobia were cultured in alternating lines on the agarmedium previously described; the legume extracts used in the medium werederived from the aerial parts of the kidney bean and of the garden pea,respectively. The colonies formed by R. phaseoli had a characteristicdark brown color whereas the colonies formed by R. leguminosarum had acharacteristic golden yellow.

The Rhizobium F₁ transconjugant derived from the milky white colonieswas cleaned and cultured as described in alternating lines with a strainof R. cowpea leucaena which was isolated from the tropical tree Leucaenaleucocephala. The legume extracts used in the medium were derived fromthe aerial parts of the kidney bean, the garden pea and the leaves ofLeucaena leucocephala. The colonies formed by the F₁ transconjugant weremilky white whereas the colonies formed by R. cowpea leucaena had acharacteristic greyish brown color.

The Rhizobium F₂ transconjugant derived from the snowy white coloniesobtained in between the streaks, herein called Rhizobium hordei, wascleaned and isolated as described. The F₂ transconjugant was used tonodulate barley.

8.1.1. CHARACTERIZATION OF RHIZOBIUM HORDEI

Plasmid DNA of the three parent strains, the F₁ transconjugant, and theF₂ transconjugant (R. hordei) were isolated using a modification of themethod of Hirsch et al. (1980, J. Gen. Microbiol. 120: 403-412) whichinvolved lysing the Rhizobia by an overnight incubation at 4° C. in 40%SDS (sodium dodecyl sulfate), isolating the plasmid DNA which wasseparated by electrophoresis in 0.7% agarose gels. Results of suchanalysis revealed that the R. hordei had additional plasmids of lowmolecular weight that were not observed in the three parent Rhizobia orthe F₁ transconjugant.

8.2. NODULATION OF BARLEY WITH RHIZOBIUM HORDEI

The Rhizobium hordei was used to nodulate four types of barley: Hasso,Cerise, Igri and Harry. The barley seeds were treated by immersion threetimes in the aqueous solution previously described containing 3% calciumsulphate and up to approximately 10% legume extracts which were used toproduce the R. hordei; i.e., the legume extracts used to coat the seedswere derived from the aerial parts of the kidney bean, garden pea andthe leaves of Leucaena leucocephala. After each immersion the seeds wereair-dried and sown as described below.

8.2.1. LABORATORY STUDY

The experimental protocol previously described was carried out for eachtype of barley used; i.e., 4 to 5 coated seeds were sown per one litercontainer filled with "Fibo"-clinkers and allowed to germinate. Eachtype of barley was divided into the following three groups; (a) +R-N,the seedlings of which were watered with a 50 ml suspension of R. hordeiprepared as previously described, followed by weekly watering with thenon-nitrogenous fertilizer; (b) -R+N, the seedlings of which received noR. hordei but were watered with the inorganic nitrogen fertilizer; and(c) -R-N, the seedlings of which received neither the R. hordei nor theinorganic nitrogen fertilizer but instead were watered with thenon-nitrogenous fertilizer. The plants treated with R. hordei developedroot nodules in 8 to 10 weeks. The plants in each container were thinnedto three plants each and ten containers were used in each group foranalysis of dry mass and nitrogen content.

8.3. ANALYSIS OF DRY MASS, NITROGEN CONTENT AND PROTEIN CONTENT OF THENODULATED BARLEY

The analytical methods described in Section 6.4. were used tocharacterize the nodulated barley plants.

8.3.1. RESULTS OF LABORATORY STUDY

The plants were harvested from the containers at 59, 71, 85, 108 and 128days after sowing. The dry weight per plant and nitrogen content perplant (Kjeldahl analysis) were analyzed as previously described.

The results are shown in FIG. 2 in which the dry weight per plant (A)and nitrogen content per plant (B) are plotted over the number of daysafter sowing.

In order to facilitate the understanding of the figure, the followinginformation about the average dry matter weight and average contents ofnitrogen of each barley type is helpful:

    ______________________________________                                                   Dry matter    Nitrogen                                             Barley     grams per 100 seeds                                                                         mg per seed                                          ______________________________________                                        Hasso      3.922         0.732                                                Cerise     4.217         0.792                                                Igri       3.932         0.775                                                Harry      5.068         0.826                                                ______________________________________                                    

The number of harvested plants at the various time points were:

    ______________________________________                                                 Number of Plants Harvested                                                    Days After Sowing                                                    Plant Group                                                                              59        71    85     108  128                                    ______________________________________                                        Trial One:                                                                    +R-N       23        23    27     31   37                                     -R+N       17        14    30     23   40                                     -R-N       14        18    28     20   32                                     Trial Two                                                                     +R-N        8         8     9     10   12                                     -R+N        6         5    10      8   13                                     -R-N        5         6     9      7   11                                     ______________________________________                                    

The results shown in FIG. 2 demonstrates that during the growth periodthe Rhizobium treated plants had a higher content of nitrogen and drymatter than the plants which were fertilized with nitrogen. At thetemperature of the experiment the dry content of the +R-N plants wasalmost 18-22% higher than that of the -R+N plants and both of thesecategories had a higher weight than the -R-N barley plants treated withnon-nitrogenous fertilizer.

8.4. ¹⁵ ENRICHMENT IN NODULATED BARLEY

A barley plant was incubated in the presence of atmospheric ¹⁵ N afterwhich parts of the plant were assayed for ¹⁵ N content as an indicationof nitrogen fixation. More particularly, a barley plant (age 95 days)with small nodules was placed in a chamber so that the roots wereincubated in the presence of atmospheric ¹⁵ N for 23 hours. Theatmosphere in the root chamber contained 80% N₂ (in which ¹⁵ N was 12.85At%) and 20% O₂. After the incubation period, samples were analyzed for¹⁵ N content and the total nitrogen content of the plant was determinedusing the Kjeldahl method. Results are shown in Table III below.

                  TABLE III                                                       ______________________________________                                                 NITROGEN CONTENT OF NODULATED                                                 BARLEY PARTS                                                         Plant      Dry Wt      N       Total N                                        Part       (g)         (mg/g)  (mg)                                           ______________________________________                                        Roots      0.448       8.9     3.99                                           Shoots     1.017       7.5     7.63                                           Whole Plant                                                                              1.465        7.93   11.62                                          ______________________________________                                                 .sup.15 N CONTENT OF NODULATED                                                BARLEY PARTS                                                                    Number of                                                                     Samples             At % .sup.15 N*                                ______________________________________                                        Roots      4                   0.450 ± 0.011                               Shoots     3                   0.392 ± 0.003                               ______________________________________                                         *.sup.15 N content in atmospheric air is about 0.370 At %.               

The data in Table III demonstrate that both the roots and shoots of thenodulated barley plant contain significantly more ¹⁵ N than does theatmosphere. These results indicate that nitrogen fixation took place inthe nodulated barley plant.

8.5. MORPHOLOGY OF THE BARLEY NODULES

Electron microscopy was performed on some of the large nodules; thisinvolved the examination of ultrathin cross-sections stained with uranylacetate and lead citrate. The cellular organization of the barleynodules was the same as the organization reported by Newcomb (1976, CanJ. Bot. 54: 2163-2186) for pea nodules; i.e., the vascular bundles werefound in the peripheral cortex while the central part of the nodule wasoccupied by bacteroid-filled plant cells.

A further study involving light microscopy and electron microscopy (bothscanning and transmission) of the surface of small barley nodules andthe surface and transections of large barley nodules was conducted. Themorphologies observed were compared to those of pea, white clover andsoybean. Small barley nodules were visible when the plants reached about50 days growth and formed on approximately 75% of the plants (thisobservation was made for wheat plants as well). Large barley noduleswere observed at 89 to 110 days growth. The large barley nodules wererare but unmistakable; these measured approximately 2 to 4 mm in lengthand 1 to 2 mm in diameter and occurred on the main root at a location ofabout 2 to 6 cm below the position of the old seed. Eleven of thirteenlarge nodules which were dissected were red-brown in color inside, i.e.,the same coloration as leghemoglobin in legume nodules at about 20-35days old. Of the remaining two large nodules dissected, one was whiteand appeared as a young immature legume nodule and the other was green,just as a senescing legume nodule. Possibly, barley infection occurs intwo stages: in the initial stage the R. hordei enters the root but doesnot elicit a nodule response; in the next stage formation of the nodulebegins after a lag-period.

8.6. ANTIBIOTIC RESISTANCE OF REISOLATED BARLEY BACTEROIDS

Bacteria were reisolated from the large barley nodules and tested forantibiotic resistance. The reisolated bacteria were resistant tospectinomycin dihydrochloride at a concentration of up to 400 ug/ml. TheR. hordei F₂ transconjugant originally used to infect the barley plants(i.e., the inoculant) exhibits the same resistance. Both the inoculantand the reisolate formed white opaque colonies when grown on ordinaryyeast-mannitol agar.

One of the parents of the R. hordei F₂ transconjugant is also resistantto spectinomycin dihydrochloride to the same concentration. This parentis the R. leguminosarum strain MAI which forms light yellow opaquecolonies when grown on ordinary yeast-mannitol agar. The spectinomycinresistance gene is not resident on the Sym plasmid therefore, the R.hordei F₂ transconjugant appears to be a hybrid which contains anon-symbiotic plasmid or the main chromosome of the parent R.lequminosarum MAl. However, this does not exclude the possibility thatthe R. hordei F₂ transconjugant may also harbor the Sym plasmid of MAl.

9. EXAMPLE: RHIZOBIUM SORGHI WHICH NODULATES SORGHUM

Rhizobium sorghi which nodulates sorghum, was produced according to themethod described in Section 6 by first crossing R. lupini with R. cowpealeucaena to produce the F₁ transconjugant which was then crossed with R.meliloti. The R. sorghi thus produced was used to nodulate three typesof sorghum: Safra, Dabar and Feterita.

9.1. PREPARATION OF RHIZO

For the production of R. sorghi the following parent Rhizobia were usedto produce the F₁ transconjugant milky white colonies: (a) R. lupini,isolated from lupine found in central Jutland; and (b) R. cowpealeucaena, isolated from the tropical tree Leucaena leucocephala(belonging to the family of Fabaceae) from Papua, New Guinea.

The parent Rhizobia were cultured in alternating lines on the agarmedium previously described; the legume extracts used in the medium werederived from lupine and from the leaves of the Leucaena leucocephala,respectively. The colonies formed by R. lupini had a characteristiclight yellow color whereas the colonies formed by R. cowpea leucaena hada characteristic greyish brown color.

The Rhizobium F₁ transconjugant derived from the milky white colonieswas cleaned and cultured as described in alternating lines with a strainof R. meliloti which was isolated from alfalfa found in Stahr. Thelegume extracts used in the medium were derived from the lupine, theleaves of Leucaena leucocephala and the alfalfa. The colonies formed bythe F₁ transconjugant were milky white whereas the colonies formed by R.meliloti had a characteristic yellowish brown color.

The Rhizobium F₂ transconjuqant derived from the snowy white coloniesobtained in between the streaks, herein called Rhizobium sorghi wascleaned and isolated as described. The F₂ transconjugant was used tonodulate sorghum.

9.2. NODULATION OF SORGHUM WITH RHIZOBIUM SORGHI

The Rhizobium sorghi was used to nodulate three types of sorghum: Safra,Dabar and Feterita. The sorghum seeds were treated by immersion threetimes in the aqueous solution previously described containing 3% calciumsulphate and up to approximately 10% legume extracts which were used toproduce the R. sorghi; i.e., the legume extracts used to coat the seedswere derived from the lupine, the leaves of Leucaena leucocephala, andthe alfalfa. After each immersion the seeds were air-dried and sown asdescribed below.

9.2.1. LABORATORY STUDY

The experimental protocol previously described was carried out forSafra, Dabar and Feterita sorghum i.e., 4 to 5 coated seeds were sownper one liter container filled with "Fibo"-clinkers and allowed togerminate. Each type of sorghum was divided into the following threegroups: (a) +R-N, the seedlings of which were watered with a 50 mlsuspension of R. sorghi prepared as previously described, followed byweekly watering with the non-nitrogenous fertilizer; (b) -R+N, theseedlings of which received no R. sorghi but were watered with theinorganic nitrogen fertilizer; and (c) -R-N, the seedlings of whichreceived neither the R. sorghi nor the inorganic nitrogen fertilizer butinstead were watered with the non-nitrogenous fertilizer. The plantstreated with R. sorghi developed root nodules in 10 weeks. The plants ineach container were thinned to three plants and ten containers were usedin each group for analysis.

9.2 2. FIELD STUDY

For the field study, the coated sorghum seeds (the Feterita strain) weresown as described in Section 6.3.2. The seeds of lot (a) +R-N weretreated as described with a suspension of R. sorghi and were wateredwith non-nitrogenous fertilizer; those of lot (b) -R+N, were not treatedwith R. sorghi but were watered with the inorganic nitrogen fertilizer;and those of lot (c) -R-N, were not treated with either R. sorghi or theinorganic nitrogen fertilizer, and were simply watered with thenon-nitrogenous fertilizer.

9.3. ANALYSIS OF DRY MASS, NITROGEN CONTENT AND PROTEIN CONTENT OF THENODULATED SORGHUM

The analytical methods described in Section 6.4. were used tocharacterize the nodulated sorghum plants.

9.3.1. RESULTS OF LABORATORY STUDY

The plants were harvested at from 76 to 152 days after sowing. The dryweight per plant and nitrogen content per plant (Kjeldahl analysis) wereanalyzed as previously described.

The following information about the sorghum average dry matter weightand average contents of nitrogen is helpful:

    ______________________________________                                                    Dry matter    % Nitrogen                                          Sorghum     grams per 100 seeds                                                                         per seed                                            ______________________________________                                        Safra       3.724         2.32                                                Dabar       2.944         2.17                                                Feterita    3.840         1.42                                                ______________________________________                                    

The number of harvested plants at the various time points were:

    ______________________________________                                                   Total Number of Plants Harvested at                                Plant Group                                                                              76 to 152 Days After Sowing                                        ______________________________________                                        +R-N       121                                                                -R+N       62                                                                 -R-N       70                                                                 ______________________________________                                    

9.3.2. RESULTS OF FIELD STUDY

The sorghum plants in the field study were harvested after one season ofgrowth and the protein content per plant as well as the amino acidcomposition were analyzed as previously described. The results shown inTable IV clearly demonstrate that the plants treated with R. sorghi(+R-N) had a higher protein content than either the group treated withnitrogenous fertilizer (-R+N) or the untreated group (-R-N).

The analysis of the amino acid composition demonstrated that therelative amino acid composition of the Rhizobium treated plants is aboutthe same as that of the nitrogen fertilized plants; however tryptophanand leucine seem to be elevated in the Rhizobium treated plants.

                  TABLE IV                                                        ______________________________________                                        PROTEIN CONTENT OF SORGHUM                                                                 Protein Content of Seeds                                         Plant Group  per Plant                                                        ______________________________________                                        +R-N         34-52%                                                           -R+N         16%                                                              -R-N          4%*                                                             ______________________________________                                         * Since the -R-N group died after 14 weeks, no seed could be obtained. Th     nitrogen and protein content of the plants were determined as described. 

10. EXAMPLE: RHIZOBIUM ORYZAE WHICH NODULATES RICE

Rhizobium oryzae, which nodulates rice, was produced according to themethod described in Section 6 by first crossing R. meliloti with R.cowpea leucaena to produce the F₁ transconjugant which was then crossedwith R. trifoli. The R. oryzae thus produced was used to nodulate rice.

10.1. PREPARATION OF RHIZOBIUM ORYZAE

For the production of R. oryzae the following parent Rhizobia were usedto produce the F₁ transconjugant milky white colonies: (a) R. meliloti,isolated from alfalfa found in the vacinity of Victoria; and (b) R.cowpea leucaena, isolated from the tropical tree Leucaena leucocephala(belonging to the family of Fabaceae) from Papua, New Guinea.

The parent Rhizobia were cultured in alternating lines on the agarmedium previously described; the legume extracts used in the medium werederived from the alfalfa and from the leaves of the Leucaenaleucocephala, respectively. The colonies formed by R. meliloti had acharacteristic yellowish brown color whereas the colonies formed by R.cowpea leucaena had a characteristic greyish brown color.

The Rhizobium F₁ transconjugant derived from the milky white colonieswas cleaned and cultured as described in alternating lines with a strainof R. trifoli which was isolated from red clover, found in Randers. Thelegume extracts used in the medium were derived from the alfalfa, theleaves of Leucaena leucocephala and the red clover. The colonies formedby the F₁ transconjugant were milky white whereas the colonies formed byR. trifoli had a characteristic light brown color.

The Rhizobium F₂ transconjugant derived from the snowy white coloniesobtained in between the streaks, herein called Rhizobium oryzae wascleaned and isolated as described; the F₂ transconjugant was used tonodulate rice.

10.2. NODULATION OF RICE WITH RHIZOBIUM ORYZAE

The rice seeds were treated as described by immersion three times in theaqueous solution previously described containing 3% calcium sulphate andup to approximately 10% legume extracts which were used to produce theR. oryzae; i.e., the legume extracts used to coat the seeds were derivedfrom the alfalfa, the leaves of Leucaena leucocephala and the redclover. After each immersion the seeds were air-dried and sown asdescribed below.

10.2.1. FIELD STUDY

For the Field study, the coated rice seeds were sown as described inSection 6.3.2. The seeds of lot (a) +R-N, were treated as described witha suspension of R. oryzae and were watered with non-nitrogenousfertilizer; those of lot (b) -R+N, were not treated with R. oryzae butwere watered with the inorganic nitrogen fertilizer; and those of lot(c) -R-N, were not treated with either R. oryzae or the inorganicnitrogen fertilizer, and were simply watered with the non-nitrogenousfertilizer.

10.3. ANALYSIS OF PROTEIN AND NITROGEN CONTENT OF THE NODULATED RICE

The analytical methods described in Section 6.4. were used tocharacterize the nodulated rice plants.

10.3.1. RESULTS OF FIELD STUDY

The plants were harvested every 4 1/2 months over a 2 1/2 year period (2crops per year) and the protein content per plant was analyzed aspreviously described.

The results shown in Table V clearly demonstrate that the rice plantstreated with R. oryzae (+R-N) had a higher protein content than eitherthe group treated with nitrogenous fertilizer (-R-N) or the untreatedgroup (-R-N).

                  TABLE V                                                         ______________________________________                                        PROTEIN CONTENT OF RICE                                                                     Protein Content                                                 Plant Group   per Plant                                                       ______________________________________                                        +R-N          12.0-18%                                                        -R+N          1.5-3%                                                          -R-N          0.5-1%                                                          ______________________________________                                    

11. EXAMPLE: RHIZOBIUM AS A NITROGEN FERTILIZER FOR EUCALYPTUS

A positive effect of Rhizobium as a nitrogen fertilizer was observedwith Eucalyptus (family Myrtaceae), a plant species outside the grassfamily. Of the two species tested, E. globulus responded very clearly inincreased biomass, however, nodules were not observed.

11.1. PREPARATION OF RHIZOBIUM EUl

Rhizobium eul which has the beneficial effect on Eucalyptus was producedaccording to the method in Section 6 by first crossing R. cowpealeucaena with R. leguminosarum to produce the F₁ transconjugant whichwas then crossed with R. meliloti. The F₂ transconjugant thus produced,R. eul, was used to treat two species of Eucalyptus. This Rhizobiumstrain does not infect any other plant host described in the examplesherein.

11.2. TREATMENT OF EUCALYPTUS WITH RHIZOBIUM EUl

Eucalyptus seeds were pre-germinated, as previously described forcereals, in a legume plant extract of pea, beans, lupin, clover, alfalfaand leucaena. The treated seeds were sown in coarse sand and untreatedseeds were sown as a control.

After germination, the pretreated seedlings were watered withnon-nitrogenous fertilizer and a suspension of one of six differentRhizobium F₂ transconjugants (+R-N). After germination, one group ofuntreated seedlings was watered with a nitrogenous fertilizer (-R+N) andanother group received non-nitrogenous fertilizer (-R-N). Four weekslater the seedlings treated with five of the six Rhizobia strains hadall died; however, those treated with Rhizobium eul survived. Thesurviving plants were transferred individually to six liter pots withsoil and a top layer of "Fibo"-clinkers to retain moisture in the potand prevent growth of fungi. The bottom of the pots were perforated sothat excess water could run out, but the amount of non-nitrogenousfertilizer given was adjusted so that no standing water or bacterialcontamination was possible. There were 28 pots in all; 14 were +R-N and14 were -R+N. The 14 +R-N were re-inoculated with Rhizobium eul.

After 4 months growth, the +R-N eucalyptus plants had 2 to 3 times asmuch biomass as the -R+N plants. Examination of one +R-N plant did notreveal nodules; however, the detection of small nodules is verydifficult when the roots grow in soil because soil particles adhere tothe roots and discolor the fine roots. Despite the inability to detectnodules in this +R-N Eucalyptus plant, the results indicate that theincreased biomass is due to rhizobial nitrogen fixation.

12. EXAMPLE: RHIZOBIUM Rl WHICH NODULATES BRASSICAS

A member of the Brassicas, another plant outside the grass family, alsoresponded positively to Rhizobium treatment. Treatment of rape (Brassicanapus) with Rhizobium Rl resulted in plant growth and the appearance ofnodules.

12.1. PREPARATION OF RHIZOBIUM Rl

Rhizobium Rl which nodulates rape, was produced according to the methoddescribed in Section 6 by first crossing R. phaseoli with R. cowpealeucaena to produce the F₁ transconjugant which was then crossed with R.trifoli.

12.2. TREATMENT OF RAPE WITH RHIZOBIUM Rl

The rape seeds were pregerminated, as previously described for cereals,in a legume plant extract of bean, leucaena, and clover. The treatedseeds were sown in two liter containers and after germination one groupwas fertilized normally (-R+N), another group was watered withnon-nitrogenous fertilizer only (-R-N) and a third group which was alsowatered with non-nitrogenous fertilizer, was treated with six differentstrains of Rhizobia (+R-N). After 3 1/2 months the -R-N plants stillonly had the three first juvenile leaves and the +R-N plants treatedwith three Rhizobia strains designated R4, R5 and R6 were just as poor.However, one of the Rhizobia transconjugants, namely strain Rl, wasbeneficial. The plant treated with Rhizobium Rl had almost the same sizeand pods as the largest -R+N plant, and small root nodules were visible.

13. EXAMPLE: TOTAL NITROGEN ANALYSIS OF NODULATED NON-LEGUMES

To demonstrate that the nodules elicited by the Rhizobia transformantsof the invention in non-legumes were active in nitrogen fixation,Kjeldahl analyses of plant organic nitrogen contents were made.

13.1 MATERIALS AND METHODS

Each of the non-legumes tested were divided into three groups: (a) onewas treated with the Rhizobium transconjugant and a non-nitrogenousfertilizer (+R-N); (b) the second group was treated with nitrogenousfertilizer without the Rhizobium transconjugant (-R+N); and (c) thethird group was treated with neither the Rhizobium transconjugant northe nitrogenous fertilizer (-R-N). These group were grown underotherwise identical conditions in the growth house and harvested anumber of times throughout the growing period until seeds were mature.Three to five plants were grown in each container and at harvest theirshoots were combined. One sample was taken from this material andanalyzed for total organic nitrogen. The results presented in thesubsections below demonstrate that the nodulated non-legume plants wereable to fix nitrogen.

13.2. WHEAT AND BARLEY PLANTS

Two varieties of wheat, Cornette and Ralle, were nodulated usingRhizobium tritici as described previously in Section 7. The nitrogencontent, expressed as mg nitrogen per plant top and the dry weight ofthe plant top were assayed. Results are presented in Table VI, below.

                  TABLE VI                                                        ______________________________________                                        N CONTENT AND DRY WEIGHT OF                                                   NODULATED WHEAT                                                                    mg/     +R-N        -R+N      -R-N                                       Days Plant*  (n = 4)     (n = 2)   (n = 2)                                    ______________________________________                                               TRIAL ONE                                                              30   DW      56.05 ± 28.5                                                                           90.89 ± 26.3                                                                         39.69 ± 6.3                                  N       1.14 ± 0.5                                                                             2.67 ± 0.6                                                                           0.51 ± 0                                50   DW      120.49 ± 27.8                                                                          257.56 ± 62.3                                                                        82.61 ± 13.6                                 N       2.26 ± 0.3                                                                             3.99 ± 1.4                                                                           0.66 ± 0.3                              64   DW      244.78 ± 21.8                                                                          331.99 ± 5.8                                                                         72.21 ± 8.7                                  N       3.03 ± 0.3                                                                             5.01 ± 0.4                                                                           0.44 ± 0.1                              76   DW      314.38 ± 112.5                                                                         400.64 ± 83.2                                                                        117.53 ± 44.6                                N       4.28 ± 1.3                                                                             6.56 ± 2.1                                                                           0.69 ± 0.2                              97   DW      431.47 ± 68.5                                                                          666.71 ± 60.3                                                                        117.0 ± 54.2                                 N       5.19 ± 0.7                                                                             7.52 ±  0.6 ± 0.2                              115  DW      523.49 ± 38.6                                                                          668.55 ± 113.4                                                                       82.96 ± 3.2                                  N       5.98 ± 1.2                                                                             9.94 ± 3.5                                                                           0.42 ± 0.1                              124  DW      565.11 ± 23.2                                                                          700.77 ± 79.4                                                                        113.94 ± 12.3                                N       8.15 ± 0.4                                                                             11.43 ± 1.9                                                                          0.67 ± 0                                       TRIAL TWO                                                              54   DW      158.85 ± 36.3                                                                          304.91 ± 72.3                                                                        90.30 ± 12.4                                 N       3.59 ± 0.5                                                                             7.46 ± 0.8                                                                           0.51 ± 0.1                              71   DW      447.88 ± 83.1                                                                          705.64 ± 37.0                                                                        89.54 ± 23.0                                 N       6.28 ± 1.2                                                                             12.48 ± 1.9                                                                          0.54 ± 0.2                              80   DW      513.37 ± 120.7                                                                         871.57 ± 205.5                                                                       117.04 ± 2.6                                 N       7.61 ± 1.1                                                                             12.15 ± 2.4                                                                          0.71 ± 0.1                              92   DW      903.53 ± 25.6                                                                          1128.85 ± 166.4                                                                      131.18 ± 11.8                                N       11.81 ± 0.45                                                                           14.56 ± 1.89                                                                         0.72 ± 0.1                              ______________________________________                                         *DW = Dry Weight                                                              Plants in Trial One were grown under suboptimal lighting conditions.     

The results presented in Table VI indicate that the dry weight of the+R-N plants is about 82% that of the -R+N plants and that the nitrogencontent of the +R™N plants is about 80% of the -R+N plants.

Five varieties of barley, Jenny, Taarn, Lina, Grith and Triumph, werenodulated using Rhizobium hordei as described previously in Section 8.The nitrogen content, expressed as mg nitrogen per plant top and the dryweight of the plant top were assayed. Results are presented in TableVII, below.

                  TABLE VII                                                       ______________________________________                                        N CONTENT AND DRY WEIGHT OF                                                   NODULATED BARLEY                                                                   mg/     +R-N        -R+N      -R-N                                       Days Plant*  (n = 10)    (n = 5)   (n = 5)                                    ______________________________________                                               TRIAL ONE                                                              31   DW      43.85 ± 9.2                                                                            71.84 ± 21.7                                                                         19.15 ± 5.2                                  N       0.99 ± 0.2                                                                             2.15 ± 0.9                                                                           0.27 ± 0.1                              50   DW      121.53 ± 28.7                                                                          224.95 ± 44.5                                                                        35.40 ± 4.2                                  N       2.56 ± 0.5                                                                             4.03 ± 1.3                                                                           0.41 ± 0.1                              64   DW      198.41 ± 50.7                                                                          269.92 ± 26.8                                                                        41.87 ± 7.1                                  N       2.61 ± 0.5                                                                             4.05 ± 1.1                                                                           0.35 ± 0.1                              76   DW      301.04 ± 56.1                                                                          296.76 ± 76.9                                                                        49.58 ± 4.8                                  N       4.09 ± 0.6                                                                             5.22 ± 2.9                                                                           0.61 ± 0.3                              96   DW      499.23 ± 106.9                                                                         506.33 ± 106.5                                                                       55.19 ± 15.7                                 N       5.34 ± 1.4                                                                             9.90 ± 0.35 ± 0.1                              123  DW      535.47 ± 123.7                                                                         556.38 ± 124.3                                                                       64.10 ± 11.5                                         5.55 ± 1.1                                                                             10.34 ± 3.7                                                                          0.44 ± 0.1                                     TRIAL TWO                                                              54   DW      118.89 ± 20.6                                                                          264.84 ± 38.4                                                                        49.76 ± 11.8                                 N       3.30 ± 0.4                                                                             7.78 ± 0.7                                                                           0.45 ± 0.1                              71   DW      313.06 ± 46.1                                                                          405.53 ± 120.8                                                                       53.49 ± 9.9                                  N       5.81 ± 0.6                                                                              .78 ± 0.8                                                                           0.41 ± 0.1                              80   DW      711.29 ± 279.6                                                                         617.75 ± 94.3                                                                        68.42 ± 2.6                                  N       7.85 ± 1.2                                                                             10.26 ± 2.4                                                                          0.53 ± 0.1                              92   DW      802.30 ± 147.3                                                                         837.08 ± 196.4                                                                       57.07 ± 19.5                                 N       10.08 ± 1.6                                                                            11.99 ± 1.6                                                                          0.46 ± 0.2                              ______________________________________                                         *DW = Dry Weight.                                                        

The barley plants nodulated with Rhizobium grow to the same size and dryweight as the plants treated with normal nitrogenous fertilizer. Thenitrogen content of the +R-N barley is about 83% that of the -R+Nplants, and is 20 times the nitrogen content of the -R-N plants. Thenitrogen assayed in the -R-N plants is derived from the seed and doesnot increase during the growth period.

Notably, the effect of the Rhizobium transconjugants as a substitute fornitrogen fertilizer is due to the action of small nodules which werepresent on 75% of the plants. Because 3 to 5 plants were grown percontainer, it was not possible to separate the roots and analyze onlythe nodulated plants. It is contemplated that nodulation of 100% of theplants and the development of large nodules will function even moreeffectively.

To eliminate the possibility that the +R-N nodulated non-leguminousplants may use the Rhizobia inoculum or bacteria that may laterproliferate in the containers as a source of nitrogen, the followingexperiment was performed: the Cornette variety of wheat and the Taarnvariety of barley were each inoculated with R. lequminosarum strain MAlusing the same procedures, amounts and conditions described forinoculating these nonlegume plants with the Rhizobium transconjugant, R.tritici or R. hordei, respectively. The plants inoculated with R.leguminosarum were watered with non-nitrogenous fertilizer and theaccumulation of nitrogen was compared to that of -R-N plants. Neitherthe plants inoculated with R. leguminosarum nor the -R-N plantsaccumulated nitrogen. The conclusion drawn is that a nonspecificbacterial inoculum, as such, does not supply nitrogen fertilizer to theplants. By contrast, the nitrogen accumulation shown in Tables VII andVIII which occurs in response to nodulation with R. tritici or R.hordei, respectively, must be due to nitrogen fixation.

13.3. SORGHUM AND RICE PLANTS

Three varieties of sorghum, Dabar, Safra and Feterita were nodulatedusing Rhizobium sorghi as described previously in Section 9. Thenitrogen content, expressed as mg nitrogen per plant top and the dryweight of the plant top were assayed. Results are presented in TableVIII below.

                  TABLE VIII                                                      ______________________________________                                        N CONTENT AND DRY WEIGHT OF                                                   NODULATED SORGHUM                                                                  mg/     +R-N        -R+N      -R-N                                       Days Plant*  (n = 6)     (n = 3)   (n = 3)                                    ______________________________________                                        30   DW      38.83 ± 10.1                                                                           49.60 ± 22.5                                                                         25.60 ± 4.2                                  N       0.40 ± 0.1                                                                             1.42 ± 0.7                                                                           0.26 ± 0                                59   DW      102.93 ± 15.2                                                                          329.51 ± 87.7                                                                        42.84 ± 8.0                                  N       1.00 ± 0.2                                                                             5.93 ± 2.3                                                                           0.31 ± 0                                80   DW      179.84 ± 62.2                                                                          602.34 ± 44.9                                                                        43.77 ± 21.5                                 N       2.00 ± 0.8                                                                             9.86 ± 2.7                                                                           0.31 ± 0                                101  DW      439.83 ± 95.8                                                                          576.18 ± 75.4                                                                        53.71 ± 12.4                                 N       4.40 ± 0.9                                                                             11.02 ± 0.9                                                                          0.31 ± 0                                125  DW      1242.94 ± 391.4                                                                        1198.99 ± 596.0                                                                      56.76 ± 7.8                                  N       11.65 ± 2.4                                                                            12.44 ± 6.8                                                                          0.27 ± 0                                ______________________________________                                         *DW = Dry Weight                                                         

An M-201 strain of rice was nodulated using Rhizobium oryzae asdescribed previously in Section 10. The nitrogen content expressed as mgnitrogen per plant top and the dry weight of the plant top were assayed.Results are presented in Table IX below.

                  TABLE IX                                                        ______________________________________                                        N CONTENT AND DRY WEIGHT OF                                                   NODULATED BARLEY                                                                      mg/                                                                   Days    Plant*  +R-N        -R+N   -R-N                                       ______________________________________                                        62      DW      32.28       48.87  23.44                                              N       0.32        1.10   0.16                                       74      DW      48.04       94.73  21.63                                              N       1.15        2.65   0.16                                       98      DW      107.47      115.40 29.9                                               N       2.91        3.45   0.36                                       128     DW      174.89      130.83 22.16                                              N       5.16        4.33   0.38                                       ______________________________________                                         *DW = Dry Weight                                                         

The results indicate that the dry weight accumulation of the +R-N plantsinitially lags behind that of the -R+N plants; however, by the end ofthe experimental period, the dry matter accumulated by the +R-N plantsequals, and may exceed that of the -R+N plans. The accumulation ofnitrogen in the +R-N plants and the -R+N plants appears to be about thesame.

13.4. RAPE

Two varieties of rape, Hanna and Topas, were nodulated using RhizobiumRl as described previously in Section 12. The nitrogen content,expressed as mg nitrogen per plant top and the dry weight of the planttop were assayed. Results are presented in Table X below.

                  TABLE X                                                         ______________________________________                                        N CONTENT AND DRY WEIGHT OF                                                   NODULATED BARLEY                                                                   mg/                                                                      Days Plant*  +R-N        -R+N      -R-N                                       ______________________________________                                        47   DW      67.25 ± 17.4                                                                           344.91 ± 31.6                                                                        9.71 ± 0.2                                   N       0.89 ± 0.3                                                                             6.71 ± 0.1                                                                           0.22 ± 0.2                              59   DW      99.40 ± 14.3                                                                           477.07 ± 62.8                                                                        13.90 ± 4.5                                  N       1.64 ± 0.4                                                                             7.03 ± 0.2                                                                           0.11 ± 0.1                              73   DW      248.56 ± 24.3                                                                          766.47 ± 72.7                                                                        14.92 ± 9.9                                  N       2.00 ± 0.7                                                                             10.25 ± 1.9                                                                           0.10 ± 0.04                            98   DW      424.80 ± 72.5                                                                          704.74 ±                                                                             12.11 ± 7.9                                  N       3.42 ± 1.1                                                                             17.66 ± 5.1                                                                           0.10 ± 0.05                            110  DW      547.37 ± 22.2                                                                          1049.77 ± 388.6                                                                      17.86 ± 12.1                                 N       5.73 ± 1.9                                                                             24.33 ± 7.9                                                                          0.26 ± 0.2                              129  DW      1013.59 ± 241.8                                                                        1955.60 ± 330.0                                                                      15.39 ± 6.0                                  N       10.24 ± 0.9                                                                            33.21 ± 8.6                                                                           0.16 ± 0.09                            147  DW      2231.55     ND        ND                                              N       16.66       ND        ND                                         ______________________________________                                         *DW = Dry Weight                                                              n = 4 samples                                                                 ND = no data.                                                            

The results indicate that the +R-N plants accumulated about 50% of thedry weight accumulated by the -R+N plants and the nitrogen content ofthe +R-N plants was about 30% that of the =R+N plants. Although theaccumulation was lower than that of the nitrogen fertilized plants, thesmall nodules observed in the rape plant account for the survival of thenodulated rape plants grown in the absence of nitrogenous fertilizer(compare results for +R-N to those for -R-N in which neither dry weightnor nitrogen was accumulated).

14. DEPOSIT OF MICROORGANISMS

The following Rhizobium strains have been deposited with the AmericanType Culture Collection (ATCC), Rockville, Md. and have been assignedthe following accession numbers:

    ______________________________________                                        Rhizobium     Accession No.                                                   ______________________________________                                        R. tritici    53,407                                                          R. hordei     53,404                                                          R. sorghi     53,405                                                          R. oryzae     53,406                                                          R. R1         53,563                                                          R. eu1        53,562                                                          ______________________________________                                    

The present invention is not to be limited in scope by themicroorganisms deposited since the deposited embodiment is intended as asingle illustration of one aspect of the invention and anymicroorganisms which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures Such modifications are intended to fall within the scope of theappended claims.

What is claimed is:
 1. A Rhizobium transconjugant which fixes nitrogenin wheat plants as deposited with the ATCC and assigned accession number53,407.
 2. A Rhizobium transconjugant which fixes nitrogen in barleyplants as deposited with the ATCC and assigned accession number 53,404.3. A Rhizobium transconjugant which fixes nitrogen in sorghum plants asdeposited with the ATCC and assigned accession number 53,405.
 4. ARhizobium transconjugant which fixes nitrogen in rice plants asdeposited with the ATCC and assigned accession number 53,406.
 5. ARhizobium transconjugant which fixes nitrogen in Brassica plants asdeposited with the ATCC and assigned accession number 53,564.
 6. ARhizobium transconjugant which fixes nitrogen in the vicinity ofeucalyptus roots as deposited with the ATCC and assigned accessionnumber 53,562.
 7. The Rhizobium transconjugant of claim 1, 2, 3, 4, 5,or 6 characterized a forming snowy white colonies before enteringsymbiosis with a non-legume host partner when the transconjugant iscultured on a nutrient medium containing, in addition to nutrientsrequired for growth, an extract of each legume host partner of eachparent Rhizobium species of the Rhizobium transconjugant.
 8. TheRhizobium transconjugant of claim 1, 2, 3, 4, 5, or 6 characterized aforming grey colonies after entering symbiosis with a non-legume hostpartner when the transconjugant is cultured on a nutrient mediumcontaining, in addition to nutrients required for growth, an extract ofeach legume host partner of each parent Rhizobium species of theRhizobium transconjugant and an extract of the non-legume host partnerof the Rhizobium transconjugant.
 9. The Rhizobium transconjugant ofclaim 2 which symbiotically fixes nitrogen in the barley strain Hasso.10. The Rhizobium transconjugant of claim 2 which symbiotically fixesnitrogen in the barley strain Cerise.
 11. The Rhizobium transconjugantof claim 2 which symbiotically fixes nitrogen in the barley strainHarry.
 12. The Rhizobium transconjugant of claim 2 which symbioticallyfixes nitrogen in the barley strain Igri.
 13. The Rhizobiumtransconjugant of claim 6 which is capable of acting as a nitrogenfertilizer for a non-legume belonging to the family Myrtaceae.
 14. TheRhizobium transconjugant of claim 13 which is capable of acting as anitrogen fertilizer for Eucalyptus.
 15. A method for producing theRhizobia F₂ transconjugants of claim 1, 2, 3, 4, 5, or 6 thatsymbiotically fix nitrogen in non-legumes, comprising:(a) streaking afirst Rhizobium parent and a second Rhizobium patent in alternating rowson a solid nutrient medium containing in addition to nutrient essentialfor growth, a non-denatured shoot extract of a legume host partner ofeach parent Rhizobium; (b) culturing the Rhizobia parents at atemperature of about 18° C. to about 30° C. so that the Rhizobia parentsform colonies having a specific color; (c) selecting a Rhizobium F₁transconjugant milky-white colony that grows in between the Rhizobiaparent colonies; (d) streaking the Rhizobium F₁ transconjugant inalternating rows with a third Rhizobium parent on the nutrient medium ofstep (a) further comprising a legume shoot extract of a legume hostpartner of the third Rhizobia parent; (e) culturing the Rhizobium F₁transconjugant and the third Rhizobium parent at a temperature of about18° C. to about 30° C. so that the Rhizobia form colonies; and (f)selecting a Rhizobium F₂ transconjugant snowy white colony that grows inbetween the Rhizobia F₁ transconjugant and the third Rhizobia parent.16. The method according to claim 15 in which the third Rhizobia parentcomprises a third Rhizobia species, a second Rhizobia F₁ transconjugantor a second Rhizobia F₂ transconjugant.